Compositions and methods for modulating apolipoprotein (a) expression

ABSTRACT

Provided herein are oligomeric compounds with conjugate groups targeting apoplipoprotein (a) [apo(a)]. In certain embodiments, the apo(a) targeting oligomeric compounds are conjugated to N-Acetylgalactosamine. Also disclosed herein are conjugated oligomeric compounds targeting apo(a) for use in decreasing apo(a) to treat, prevent, or ameliorate diseases, disorders or conditions related to apo(a) and/or Lp(a). Certain diseases, disorders or conditions related to apo(a) and/or Lp(a) include inflammatory, cardiovascular and/or metabolic diseases, disorders or conditions. The conjugated oligomeric compounds disclosed herein can be used to treat such diseases, disorders or conditions in an individual in need thereof.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0250USC3SEQ_ST25.txt, created on Feb. 5, 2018, which is 432 Kb insize. The information in the electronic format of the sequence listingis incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The principle behind antisense technology is that an antisense compoundhybridizes to a target nucleic acid and modulates the amount, activity,and/or function of the target nucleic acid. For example in certaininstances, antisense compounds result in altered transcription ortranslation of a target. Such modulation of expression can be achievedby, for example, target mRNA degradation or occupancy-based inhibition.An example of modulation of RNA target function by degradation is RNaseH-based degradation of the target RNA upon hybridization with a DNA-likeantisense compound. Another example of modulation of gene expression bytarget degradation is RNA interference (RNAi). RNAi refers toantisense-mediated gene silencing through a mechanism that utilizes theRNA-induced siliencing complex (RISC). An additional example ofmodulation of RNA target function is by an occupancy-based mechanismsuch as is employed naturally by microRNA. MicroRNAs are smallnon-coding RNAs that regulate the expression of protein-coding RNAs. Thebinding of an antisense compound to a microRNA prevents that microRNAfrom binding to its messenger RNA targets, and thus interferes with thefunction of the microRNA. MicroRNA mimics can enhance native microRNAfunction. Certain antisense compounds alter splicing of pre-mRNA.Regardless of the specific mechanism, sequence-specificity makesantisense compounds attractive as tools for target validation and genefunctionalization, as well as therapeutics to selectively modulate theexpression of genes involved in the pathogenesis of diseases.

Antisense technology is an effective means for modulating the expressionof one or more specific gene products and can therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications. Chemically modified nucleosides may be incorporated intoantisense compounds to enhance one or more properties, such as nucleaseresistance, pharmacokinetics or affinity for a target nucleic acid. In1998, the antisense compound, Vitravene® (fomivirsen; developed by IsisPharmaceuticals Inc., Carlsbad, Calif.) was the first antisense drug toachieve marketing clearance from the U.S. Food and Drug Administration(FDA), and is currently a treatment of cytomegalovirus (CMV)-inducedretinitis in AIDS patients.

New chemical modifications have improved the potency and efficacy ofantisense compounds, uncovering the potential for oral delivery as wellas enhancing subcutaneous administration, decreasing potential for sideeffects, and leading to improvements in patient convenience. Chemicalmodifications increasing potency of antisense compounds allowadministration of lower doses, which reduces the potential for toxicity,as well as decreasing overall cost of therapy. Modifications increasingthe resistance to degradation result in slower clearance from the body,allowing for less frequent dosing. Different types of chemicalmodifications can be combined in one compound to further optimize thecompound's efficacy.

Lipoproteins are globular, micelle-like particles that consist of anon-polar core of acylglycerols and cholesteryl esters surrounded by anamphiphilic coating of protein, phospholipid and cholesterol.Lipoproteins have been classified into five broad categories on thebasis of their functional and physical properties: chylomicrons, verylow density lipoproteins (VLDL), intermediate density lipoproteins(IDL), low density lipoproteins (LDL), and high density lipoproteins(HDL). Chylomicrons transport dietary lipids from intestine to tissues.VLDLs, IDLs and LDLs all transport triacylglycerols and cholesterol fromthe liver to tissues. HDLs transport endogenous cholesterol from tissuesto the liver

Lipoprotein particles undergo continuous metabolic processing and havevariable properties and compositions. Lipoprotein densities increasewithout increasing particle diameter because the density of their outercoatings is less than that of the inner core. The protein components oflipoproteins are known as apolipoproteins. At least nine apolipoproteinsare distributed in significant amounts among the various humanlipoproteins.

The lipoprotein(a) [Lp(a)] particle was identified nearly 50 years agoand is comprised of a highly unique LDL particle in which oneapolipoprotein B (apoB) protein is linked via a disulfide bond to asingle apolipoprotein(a) [apo(a)] protein. The apo(a) protein shares ahigh degree of homology with plasminogen particularly within the kringleIV type 2 repetitive domain. Levels of circulating Lp(a) are inverselyproportional to the number of kringle IV type 2 variable repeats presentin the molecule and, as both alleles are co-expressed withinindividuals, can display heterozygous plasma isoform profiles (Kraft etal., Eur J Hum Genet, 1996; 4(2): 74-87). It is thought that thiskringle repeat domain in apo(a) may be responsible for itspro-thrombotic and anti-fibrinolytic properties, potentially enhancingatherosclerotic progression.

Apo(a) is transcriptionally regulated by IL-6 and in studies inrheumatoid arthritis patients treated with an IL-6 inhibitor(tocilizumab), plasma levels were reduced by 30% after 3 month treatment(Schultz et al., PLoS One 2010; 5:e14328).

Apo(a) has been shown to preferentially bind oxidized phospholipids andpotentiate vascular inflammation (Bergmark et al., J Lipid Res 2008;49:2230-2239; Tsimikas et al., Circulation. 2009; 119(13):1711-1719).

Further, studies suggest that the Lp(a) particle may also stimulateendothelial permeability, induce plasminogen activator inhibitor type-1expression and activate macrophage interleukin-8 secretion (Koschinskyand Marcovina, Curr Opin Lipidol 2004; 15:167-174). Importantly, recentgenetic association studies revealed that Lp(a) was an independent riskfactor for myocardial infarction, stroke, peripheral vascular diseaseand abdominal aortic aneurysm (Rifai et al., Clin Chem 2004; 50:1364-71;Erqou et al., JAMA 2009; 302:412-23; Kamstrup et al., Circulation 2008;117:176-84). Further, in the recent Precocious Coronary Artery Disease(PROCARDIS) study, Clarke et al. (Clarke et al., NEJM (2009) 361;2518-2528) described robust and independent associations betweencoronary heart disease and plasma Lp(a) concentrations. Additionally,Solfrizzi et al., suggested that increased serum Lp(a) may be linked toan increased risk for Alzheimer's Disease (AD) (Solfrizzi et al., JNeurol Neurosurg Psychiatry 2002, 72:732-736. Currently, in the clinicsetting, examples of indirect apo(a) inhibitors for treatingcardiovascular disease include aspirin, Niaspan, Mipomersen,Anacetrapib, Epirotirome and Lomitapide which reduce plasma Lp(a) levelsby 18%, 39%, 32%, 36%, 43% and 17%, respectively. Additionally, Lp(a)apheresis has been used in the clinic to reduce apo(a) containing Lp(a)particles.

To date, therapeutic strategies to treat cardiovascular disease bydirectly targeting apo(a) levels have been limited. Ribozymeoligonucleotides (U.S. Pat. No. 5,877,022) and antisenseoligonucleotides (WO 2005/000201; WO 2003/014397; WO2013/177468;US20040242516; U.S. Pat. Nos. 8,138,328, 8,673,632 and 7,259,150; Merkiet al., J Am Coll Cardiol 2011; 57:1611-1621; each publicationincorporated by reference in its entirety) have been developed but nonehave been approved for commercial use.

Thus, there remains a clear unmet medical need for novel agents whichcan potently and selectively reduce apo(a) levels in patients atenhanced risk for cardiovascular events due to chronically elevatedplasma Lp(a) levels.

SUMMARY OF THE INVENTION

Provided herein are compositions and methods for modulating expressionof apo(a) mRNA and protein. In certain embodiments, the apo(a) specificinhibitor decreases expression of apo(a) mRNA and protein. Providedherein are compositions and methods for modulating expression of Lp(a)levels.

In certain embodiments, the composition is an apo(a) specific inhibitor.In certain embodiments, the apo(a) specific inhibitor is a nucleic acid,protein, or small molecule. In certain embodiments, the apo(a) specificinhibitor is an antisense oligonucleotide targeting apo(a) with aconjugate. In certain embodiments, the apo(a) specific inhibitor is amodified oligonucleotide and a conjugate, wherein the modifiedoligonucleotide consists of 12 to 30 linked nucleosides and comprises anucleobase sequence comprising a portion of at least 8 contiguousnucleobases complementary to an equal length portion of nucleobases 3901to 3920 of SEQ ID NO: 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 80% complementary to SEQ ID NO: 1. Incertain embodiments, the apo(a) specific inhibitor is a modifiedoligonucleotide and a conjugate, wherein the modified oligonucleotideconsists of 12 to 30 linked nucleosides and has a nucleobase sequencecomprising at least 8, least 9, least 10, least 11, at least 12, least13, at least 14, at least 15, at least 16, least 17, least 18, least 19,or 20 contiguous nucleobases of the nucleobase sequence of SEQ ID NO:1-130, 133, 134. In certain embodiments, the apo(a) specific inhibitoris a modified oligonucleotide and a conjugate, wherein the modifiedoligonucleotide consists of 20 linked nucleosides and has a nucleobasesequence comprising at least 8 contiguous nucleobases of any of SEQ IDNO: 58, wherein the modified oligonucleotide comprises: (a) a gapsegment consisting of ten linked deoxynucleosides; (b) a 5′ wing segmentconsisting of five linked nucleosides; (c) a 3′ wing segment consistingof five linked nucleosides; and wherein the gap segment is positionedbetween the 5′ wing segment and the 3′ wing segment, wherein eachnucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar,wherein at least one internucleoside linkage is a phosphorothioatelinkage and wherein each cytosine residue is a 5-methylcytosine.

Certain embodiments provide a composition comprising a conjugatedantisense compound described herein, or a salt thereof, and apharmaceutically acceptable carrier or diluent.

In certain embodiments, the modulation of apo(a) expression occurs in acell or tissue. In certain embodiments, the modulations occur in a cellor tissue in an animal. In certain embodiments, the animal is a human.In certain embodiments, the modulation is a reduction in apo(a) mRNAlevel. In certain embodiments, the modulation is a reduction in apo(a)protein level. In certain embodiments, both apo(a) mRNA and proteinlevels are reduced. In certain embodiments, the modulation is areduction in Lp(a) level. Such reduction may occur in a time-dependentor in a dose-dependent manner.

Certain embodiments provide conjugated antisense compositions andmethods for use in therapy. Certain embodiments provide compositions andmethods for preventing, treating, delaying, slowing the progressionand/or ameliorating apo(a) related diseases, disorders, and conditions.Certain embodiments provide compositions and methods for preventing,treating, delaying, slowing the progression and/or ameliorating Lp(a)related diseases, disorders, and conditions. In certain embodiments,such diseases, disorders, and conditions are inflammatory,cardiovascular and/or metabolic diseases, disorders, and conditions. Incertain embodiments, the compositions and methods for therapy includeadministering an apo(a) specific inhibitor to an individual in needthereof. In certain embodiments, the apo(a) specific inhibitor is anucleic acid. In certain embodiments, the nucleic acid is an antisensecompound. In certain embodiments, the antisense compound is a modifiedoligonucleotide. In certain embodiments, the antisense compound is amodified oligonucleotide with a conjugate.

In certain embodiments, the present disclosure provides conjugatedantisense compounds. In certain embodiments, the present disclosureprovides conjugated antisense compounds comprising an antisenseoligonucleotide complementary to a nucleic acid transcript. In certainembodiments, the present disclosure provides methods comprisingcontacting a cell with a conjugated antisense compound comprising anantisense oligonucleotide complementary to a nucleic acid transcript. Incertain embodiments, the present disclosure provides methods comprisingcontacting a cell with a conjugated antisense compound comprising anantisense oligonucleotide and reducing the amount or activity of anucleic acid transcript in a cell.

The asialoglycoprotein receptor (ASGP-R) has been described previously.See e.g., Park et al., PNAS vol. 102, No. 47, pp 17125-17129 (2005).Such receptors are expressed on liver cells, particularly hepatocytes.Further, it has been shown that compounds comprising clusters of threeN-acetylgalactosamine (GalNAc) ligands are capable of binding to theASGP-R, resulting in uptake of the compound into the cell. See e.g.,Khorev et al., Bioorganic and Medicinal Chemistry, 16, 9, pp 5216-5231(May 2008). Accordingly, conjugates comprising such GalNAc clusters havebeen used to facilitate uptake of certain compounds into liver cells,specifically hepatocytes. For example it has been shown that certainGalNAc-containing conjugates increase activity of duplex siRNA compoundsin liver cells in vivo. In such instances, the GalNAc-containingconjugate is typically attached to the sense strand of the siRNA duplex.Since the sense strand is discarded before the antisense strandultimately hybridizes with the target nucleic acid, there is littleconcern that the conjugate will interfere with activity. Typically, theconjugate is attached to the 3′ end of the sense strand of the siRNA.See e.g., U.S. Pat. No. 8,106,022. Certain conjugate groups describedherein are more active and/or easier to synthesize than conjugate groupspreviously described.

In certain embodiments of the present invention, conjugates are attachedto single-stranded antisense compounds, including, but not limited toRNase H based antisense compounds and antisense compounds that altersplicing of a pre-mRNA target nucleic acid. In such embodiments, theconjugate should remain attached to the antisense compound long enoughto provide benefit (improved uptake into cells) but then should eitherbe cleaved, or otherwise not interfere with the subsequent stepsnecessary for activity, such as hybridization to a target nucleic acidand interaction with RNase H or enzymes associated with splicing orsplice modulation. This balance of properties is more important in thesetting of single-stranded antisense compounds than in siRNA compounds,where the conjugate may simply be attached to the sense strand.Disclosed herein are conjugated single-stranded antisense compoundshaving improved potency in liver cells in vivo compared with the sameantisense compound lacking the conjugate. Given the required balance ofproperties for these compounds such improved potency is surprising.

In certain embodiments, conjugate groups herein comprise a cleavablemoiety. As noted, without wishing to be bound by mechanism, it islogical that the conjugate should remain on the compound long enough toprovide enhancement in uptake, but after that, it is desirable for someportion or, ideally, all of the conjugate to be cleaved, releasing theparent compound (e.g., antisense compound) in its most active form. Incertain embodiments, the cleavable moiety is a cleavable nucleoside.Such embodiments take advantage of endogenous nucleases in the cell byattaching the rest of the conjugate (the cluster) to the antisenseoligonucleotide through a nucleoside via one or more cleavable bonds,such as those of a phosphodiester linkage. In certain embodiments, thecluster is bound to the cleavable nucleoside through a phosphodiesterlinkage. In certain embodiments, the cleavable nucleoside is attached tothe antisense oligonucleotide (antisense compound) by a phosphodiesterlinkage. In certain embodiments, the conjugate group may comprise two orthree cleavable nucleosides. In such embodiments, such cleavablenucleosides are linked to one another, to the antisense compound and/orto the cluster via cleavable bonds (such as those of a phosphodiesterlinkage). Certain conjugates herein do not comprise a cleavablenucleoside and instead comprise a cleavable bond. It is shown that thatsufficient cleavage of the conjugate from the oligonucleotide isprovided by at least one bond that is vulnerable to cleavage in the cell(a cleavable bond).

In certain embodiments, conjugated antisense compounds are prodrugs.Such prodrugs are administered to an animal and are ultimatelymetabolized to a more active form. For example, conjugated antisensecompounds are cleaved to remove all or part of the conjugate resultingin the active (or more active) form of the antisense compound lackingall or some of the conjugate.

In certain embodiments, conjugates are attached at the 5′ end of anoligonucleotide. Certain such 5′-conjugates are cleaved more efficientlythan counterparts having a similar conjugate group attached at the 3′end. In certain embodiments, improved activity may correlate withimproved cleavage. In certain embodiments, oligonucleotides comprising aconjugate at the 5′ end have greater efficacy than oligonucleotidescomprising a conjugate at the 3′ end (see, for example, Examples 56, 81,83, and 84). Further, 5′-attachment allows simpler oligonucleotidesynthesis. Typically, oligonucleotides are synthesized on a solidsupport in the 3′ to 5′ direction. To make a 3′-conjugatedoligonucleotide, typically one attaches a pre-conjugated 3′ nucleosideto the solid support and then builds the oligonucleotide as usual.However, attaching that conjugated nucleoside to the solid support addscomplication to the synthesis. Further, using that approach, theconjugate is then present throughout the synthesis of theoligonucleotide and can become degraded during subsequent steps or maylimit the sorts of reactions and reagents that can be used. Using thestructures and techniques described herein for 5′-conjugatedoligonucleotides, one can synthesize the oligonucleotide using standardautomated techniques and introduce the conjugate with the final(5′-most) nucleoside or after the oligonucleotide has been cleaved fromthe solid support.

In view of the art and the present disclosure, one of ordinary skill caneasily make any of the conjugates and conjugated oligonucleotidesherein. Moreover, synthesis of certain such conjugates and conjugatedoligonucleotides disclosed herein is easier and/or requires few steps,and is therefore less expensive than that of conjugates previouslydisclosed, providing advantages in manufacturing. For example, thesynthesis of certain conjugate groups consists of fewer synthetic steps,resulting in increased yield, relative to conjugate groups previouslydescribed. Conjugate groups such as GalNAc3-10 in Example 46 andGalNAc3-7 in Example 48 are much simpler than previously describedconjugates such as those described in U.S. Pat. No. 8,106,022 or7,262,177 that require assembly of more chemical intermediates.Accordingly, these and other conjugates described herein have advantagesover previously described compounds for use with any oligonucleotide,including single-stranded oligonucleotides and either strand ofdouble-stranded oligonucleotides (e.g., siRNA).

Similarly, disclosed herein are conjugate groups having only one or twoGalNAc ligands. As shown, such conjugates groups improve activity ofantisense compounds. Such compounds are much easier to prepare thanconjugates comprising three GalNAc ligands. Conjugate groups comprisingone or two GalNAc ligands may be attached to any antisense compounds,including single-stranded oligonucleotides and either strand ofdouble-stranded oligonucleotides (e.g., siRNA).

In certain embodiments, the conjugates herein do not substantially altercertain measures of tolerability. For example, it is shown herein thatconjugated antisense compounds are not more immunogenic thanunconjugated parent compounds. Since potency is improved, embodiments inwhich tolerability remains the same (or indeed even if tolerabilityworsens only slightly compared to the gains in potency) have improvedproperties for therapy.

In certain embodiments, conjugation allows one to alter antisensecompounds in ways that have less attractive consequences in the absenceof conjugation. For example, in certain embodiments, replacing one ormore phosphorothioate linkages of a fully phosphorothioate antisensecompound with phosphodiester linkages results in improvement in somemeasures of tolerability. For example, in certain instances, suchantisense compounds having one or more phosphodiester are lessimmunogenic than the same compound in which each linkage is aphosphorothioate. However, in certain instances, as shown in Example 26,that same replacement of one or more phosphorothioate linkages withphosphodiester linkages also results in reduced cellular uptake and/orloss in potency. In certain embodiments, conjugated antisense compoundsdescribed herein tolerate such change in linkages with little or no lossin uptake and potency when compared to the conjugatedfull-phosphorothioate counterpart. In fact, in certain embodiments, forexample, in Examples 44, 57, 59, and 86, oligonucleotides comprising aconjugate and at least one phosphodiester internucleoside linkageactually exhibit increased potency in vivo even relative to a fullphosphorothioate counterpart also comprising the same conjugate.Moreover, since conjugation results in substantial increases inuptake/potency a small loss in that substantial gain may be acceptableto achieve improved tolerability. Accordingly, in certain embodiments,conjugated antisense compounds comprise at least one phosphodiesterlinkage.

In certain embodiments, conjugation of antisense compounds hereinresults in increased delivery, uptake and activity in hepatocytes. Thus,more compound is delivered to liver tissue. However, in certainembodiments, that increased delivery alone does not explain the entireincrease in activity. In certain such embodiments, more compound entershepatocytes. In certain embodiments, even that increased hepatocyteuptake does not explain the entire increase in activity. In suchembodiments, productive uptake of the conjugated compound is increased.For example, as shown in Example 102, certain embodiments ofGalNAc-containing conjugates increase enrichment of antisenseoligonucleotides in hepatocytes versus non-parenchymal cells. Thisenrichment is beneficial for oligonucleotides that target genes that areexpressed in hepatocytes.

In certain embodiments, conjugated antisense compounds herein result inreduced kidney exposure. For example, as shown in Example 20, theconcentrations of antisense oligonucleotides comprising certainembodiments of GalNAc-containing conjugates are lower in the kidney thanthat of antisense oligonucleotides lacking a GalNAc-containingconjugate. This has several beneficial therapeutic implications. Fortherapeutic indications where activity in the kidney is not sought,exposure to kidney risks kidney toxicity without corresponding benefit.Moreover, high concentration in kidney typically results in loss ofcompound to the urine resulting in faster clearance. Accordingly fornon-kidney targets, kidney accumulation is undesired.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the formula:A-B-C-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In the above diagram and in similar diagrams herein, the branching group“D” branches as many times as is necessary to accommodate the number of(E-F) groups as indicated by “q”. Thus, where q=1, the formula is:A-B-C-D-E-F

where q=2, the formula is:

where q=3, the formula is:

where q=4, the formula is:

where q=5, the formula is:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

The present disclosure provides the following non-limiting numberedembodiments:

In embodiments having more than one of a particular variable (e.g., morethan one “m” or “n”), unless otherwise indicated, each such particularvariable is selected independently. Thus, for a structure having morethan one n, each n is selected independently, so they may or may not bethe same as one another.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises modified oligonucleotideISIS 494372 with a 5′-X, wherein X is a conjugate group comprisingGalNAc. In certain embodiments, the antisense compound consists ofmodified oligonucleotide ISIS 494372 with a 5′-X, wherein X is aconjugate group comprising GalNAc.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the conjugated modifiedoligonucleotide ISIS 681251. In certain embodiments, the antisensecompound consists of the conjugated modified oligonucleotide ISIS681251.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the conjugated modifiedoligonucleotide ISIS 681257. In certain embodiments, the antisensecompound consists of the conjugated modified oligonucleotide ISIS681257.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises a modified oligonucleotidewith SEQ ID NO: 58 with a 5′-GalNAc with variability in the sugar modsof the wings. In certain embodiments, the antisense compound consists ofa modified oligonucleotide with SEQ ID NO: 58 with a 5′-GalNAc withvariability in the sugar mods of the wings.

Wherein either R¹ is —OCH₂CH₂OCH₃ (MOE) and R² is H; or R¹ and R²together form a bridge, wherein R¹ is —O— and R² is —CH₂—, —CH(CH₃)—, or—CH₂CH₂—, and R¹ and R² are directly connected such that the resultingbridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

And for each pair of R³ and R⁴ on the same ring, independently for eachring: either R³ is selected from H and —OCH₂CH₂OCH₃ and R⁴ is H; or R³and R⁴ together form a bridge, wherein R³ is —O—, and R⁴ is —CH₂—,—CH(CH₃)—, or —CH₂CH₂— and R³ and R⁴ are directly connected such thatthe resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and—O—CH₂CH₂—;

And R⁵ is selected from H and —CH₃;

And Z is selected from S⁻ and O⁻.

The present disclosure provides the following non-limiting numberedembodiments:

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the disclosure. Herein, the use of thesingular includes the plural unless specifically stated otherwise. Asused herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

A. Definitions

Unless specific definitions are provided, the nomenclature used inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Certain such techniques and procedures may be foundfor example in “Carbohydrate Modifications in Antisense Research” Editedby Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,21^(st) edition, 2005; and “Antisense Drug Technology, Principles,Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press,Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratoryManual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989,which are hereby incorporated by reference for any purpose. Wherepermitted, all patents, applications, published applications and otherpublications and other data referred to throughout in the disclosure areincorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, “nucleoside” means a compound comprising a nucleobasemoiety and a sugar moiety. Nucleosides include, but are not limited to,naturally occurring nucleosides (as found in DNA and RNA) and modifiednucleosides. Nucleosides may be linked to a phosphate moiety.

As used herein, “chemical modification” means a chemical difference in acompound when compared to a naturally occurring counterpart. Chemicalmodifications of oligonucleotides include nucleoside modifications(including sugar moiety modifications and nucleobase modifications) andinternucleoside linkage modifications. In reference to anoligonucleotide, chemical modification does not include differences onlyin nucleobase sequence.

As used herein, “furanosyl” means a structure comprising a 5-memberedring comprising four carbon atoms and one oxygen atom.

As used herein, “naturally occurring sugar moiety” means a ribofuranosylas found in naturally occurring RNA or a deoxyribofuranosyl as found innaturally occurring DNA.

As used herein, “sugar moiety” means a naturally occurring sugar moietyor a modified sugar moiety of a nucleoside.

As used herein, “modified sugar moiety” means a substituted sugar moietyor a sugar surrogate.

As used herein, “substituted sugar moiety” means a furanosyl that is nota naturally occurring sugar moiety. Substituted sugar moieties include,but are not limited to furanosyls comprising substituents at the2′-position, the 3′-position, the 5′-position and/or the 4′-position.Certain substituted sugar moieties are bicyclic sugar moieties.

As used herein, “2′-substituted sugar moiety” means a furanosylcomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted sugar moiety is not a bicyclicsugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moietydoes not form a bridge to another atom of the furanosyl ring.

As used herein, “MOE” means —OCH₂CH₂OCH₃.

As used herein, “2′-F nucleoside” refers to a nucleoside comprising asugar comprising fluorine at the 2′ position. Unless otherwiseindicated, the fluorine in a 2′-F nucleoside is in the ribo position(replacing the OH of a natural ribose).

As used herein the term “sugar surrogate” means a structure that doesnot comprise a furanosyl and that is capable of replacing the naturallyoccurring sugar moiety of a nucleoside, such that the resultingnucleoside sub-units are capable of linking together and/or linking toother nucleosides to form an oligomeric compound which is capable ofhybridizing to a complementary oligomeric compound. Such structuresinclude rings comprising a different number of atoms than furanosyl(e.g., 4, 6, or 7-membered rings); replacement of the oxygen of afuranosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); orboth a change in the number of atoms and a replacement of the oxygen.Such structures may also comprise substitutions corresponding to thosedescribed for substituted sugar moieties (e.g., 6-membered carbocyclicbicyclic sugar surrogates optionally comprising additionalsubstituents). Sugar surrogates also include more complex sugarreplacements (e.g., the non-ring systems of peptide nucleic acid). Sugarsurrogates include without limitation morpholinos, cyclohexenyls andcyclohexitols.

As used herein, “bicyclic sugar moiety” means a modified sugar moietycomprising a 4 to 7 membered ring (including but not limited to afuranosyl) comprising a bridge connecting two atoms of the 4 to 7membered ring to form a second ring, resulting in a bicyclic structure.In certain embodiments, the 4 to 7 membered ring is a sugar ring. Incertain embodiments the 4 to 7 membered ring is a furanosyl. In certainsuch embodiments, the bridge connects the 2′-carbon and the 4′-carbon ofthe furanosyl.

As used herein, “nucleic acid” refers to molecules composed of monomericnucleotides. A nucleic acid includes ribonucleic acids (RNA),deoxyribonucleic acids (DNA), single-stranded nucleic acids (ssDNA),double-stranded nucleic acids (dsDNA), small interfering ribonucleicacids (siRNA), and microRNAs (miRNA). A nucleic acid may also compriseany combination of these elements in a single molecule.

As used herein, “nucleotide” means a nucleoside further comprising aphosphate linking group. As used herein, “linked nucleosides” may or maynot be linked by phosphate linkages and thus includes, but is notlimited to “linked nucleotides.” As used herein, “linked nucleosides”are nucleosides that are connected in a continuous sequence (i.e. noadditional nucleosides are present between those that are linked).

As used herein, “nucleobase” means a group of atoms that can be linkedto a sugar moiety to create a nucleoside that is capable ofincorporation into an oligonucleotide, and wherein the group of atoms iscapable of bonding with a complementary naturally occurring nucleobaseof another oligonucleotide or nucleic acid. Nucleobases may be naturallyoccurring or may be modified. As used herein, “nucleobase sequence”means the order of contiguous nucleobases independent of any sugar,linkage, or nucleobase modification.

As used herein the terms, “unmodified nucleobase” or “naturallyoccurring nucleobase” means the naturally occurring heterocyclicnucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) (including 5-methylC), and uracil (U).

As used herein, “modified nucleobase” means any nucleobase that is not anaturally occurring nucleobase.

As used herein, “modified nucleoside” means a nucleoside comprising atleast one chemical modification compared to naturally occurring RNA orDNA nucleosides. Modified nucleosides comprise a modified sugar moietyand/or a modified nucleobase.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleosidecomprising a bicyclic sugar moiety.

As used herein, “constrained ethyl nucleoside” or “cEt” means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH(CH₃)—O-2′ bridge.

As used herein, “locked nucleic acid nucleoside” or “LNA” means anucleoside comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′bridge.

As used herein, “2′-substituted nucleoside” means a nucleosidecomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted nucleoside is not a bicyclicnucleoside.

As used herein, “deoxynucleoside” means a nucleoside comprising 2′-Hfuranosyl sugar moiety, as found in naturally occurringdeoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleosidemay comprise a modified nucleobase or may comprise an RNA nucleobase(e.g., uracil).

As used herein, “oligonucleotide” means a compound comprising aplurality of linked nucleosides. In certain embodiments, anoligonucleotide comprises one or more unmodified ribonucleosides (RNA)and/or unmodified deoxyribonucleosides (DNA) and/or one or more modifiednucleosides.

As used herein “oligonucleoside” means an oligonucleotide in which noneof the internucleoside linkages contains a phosphorus atom. As usedherein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotidecomprising at least one modified nucleoside and/or at least one modifiedinternucleoside linkage.

As used herein, “linkage” or “linking group” means a group of atoms thatlink together two or more other groups of atoms.

As used herein “internucleoside linkage” means a covalent linkagebetween adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring internucleoside linkage” means a 3′to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” means anyinternucleoside linkage other than a naturally occurring internucleosidelinkage.

As used herein, “terminal internucleoside linkage” means the linkagebetween the last two nucleosides of an oligonucleotide or defined regionthereof.

As used herein, “phosphorus linking group” means a linking groupcomprising a phosphorus atom. Phosphorus linking groups include withoutlimitation groups having the formula:

wherein:

R_(a) and R_(d) are each, independently, O, S, CH₂, NH, or NJ₁ whereinJ₁ is C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

R_(b) is O or S;

R_(c) is OH, SH, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy,substituted C₁-C₆ alkoxy, amino or substituted amino; and J₁ is R_(b) isO or S.

Phosphorus linking groups include without limitation, phosphodiester,phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate,phosphorothioamidate, thionoalkylphosphonate, phosphotriesters,thionoalkylphosphotriester and boranophosphate.

As used herein, “internucleoside phosphorus linking group” means aphosphorus linking group that directly links two nucleosides.

As used herein, “non-internucleoside phosphorus linking group” means aphosphorus linking group that does not directly link two nucleosides. Incertain embodiments, a non-internucleoside phosphorus linking grouplinks a nucleoside to a group other than a nucleoside. In certainembodiments, a non-internucleoside phosphorus linking group links twogroups, neither of which is a nucleoside.

As used herein, “neutral linking group” means a linking group that isnot charged. Neutral linking groups include without limitationphosphotriesters, methylphosphonates, MMI (—CH₂—N(CH₃)—O—), amide-3(—CH₂—C(═O)—N(H)—), amide-4 (—CH₂—N(H)—C(═O)—), formacetal (—O—CH₂—O—),and thioformacetal (—S—CH₂—O—). Further neutral linking groups includenonionic linkages comprising siloxane (dialkylsiloxane), carboxylateester, carboxamide, sulfide, sulfonate ester and amides (See forexample: Carbohydrate Modifications in Antisense Research; Y. S. Sanghviand P. D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp.40-65)). Further neutral linking groups include nonionic linkagescomprising mixed N, O, S and CH₂ component parts.

As used herein, “internucleoside neutral linking group” means a neutrallinking group that directly links two nucleosides.

As used herein, “non-internucleoside neutral linking group” means aneutral linking group that does not directly link two nucleosides. Incertain embodiments, a non-internucleoside neutral linking group links anucleoside to a group other than a nucleoside. In certain embodiments, anon-internucleoside neutral linking group links two groups, neither ofwhich is a nucleoside.

As used herein, “oligomeric compound” means a polymeric structurecomprising two or more sub-structures. In certain embodiments, anoligomeric compound comprises an oligonucleotide. In certainembodiments, an oligomeric compound comprises one or more conjugategroups and/or terminal groups. In certain embodiments, an oligomericcompound consists of an oligonucleotide. Oligomeric compounds alsoinclude naturally occurring nucleic acids. In certain embodiments, anoligomeric compound comprises a backbone of one or more linked monomericsubunits where each linked monomeric subunit is directly or indirectlyattached to a heterocyclic base moiety. In certain embodiments,oligomeric compounds may also include monomeric subunits that are notlinked to a heterocyclic base moiety, thereby providing abasic sites. Incertain embodiments, the linkages joining the monomeric subunits, thesugar moieties or surrogates and the heterocyclic base moieties can beindependently modified. In certain embodiments, the linkage-sugar unit,which may or may not include a heterocyclic base, may be substitutedwith a mimetic such as the monomers in peptide nucleic acids.

As used herein, “terminal group” means one or more atom attached toeither, or both, the 3′ end or the 5′ end of an oligonucleotide. Incertain embodiments a terminal group is a conjugate group. In certainembodiments, a terminal group comprises one or more terminal groupnucleosides.

As used herein, “conjugate” or “conjugate group” means an atom or groupof atoms bound to an oligonucleotide or oligomeric compound. In general,conjugate groups modify one or more properties of the compound to whichthey are attached, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, “conjugate linker” or “linker” in the context of aconjugate group means a portion of a conjugate group comprising any atomor group of atoms and which covalently link (1) an oligonucleotide toanother portion of the conjugate group or (2) two or more portions ofthe conjugate group.

Conjugate groups are shown herein as radicals, providing a bond forforming covalent attachment to an oligomeric compound such as anantisense oligonucleotide. In certain embodiments, the point ofattachment on the oligomeric compound is the 3′-oxygen atom of the3′-hydroxyl group of the 3′ terminal nucleoside of the oligomericcompound. In certain embodiments the point of attachment on theoligomeric compound is the 5′-oxygen atom of the 5′-hydroxyl group ofthe 5′ terminal nucleoside of the oligomeric compound. In certainembodiments, the bond for forming attachment to the oligomeric compoundis a cleavable bond. In certain such embodiments, such cleavable bondconstitutes all or part of a cleavable moiety.

In certain embodiments, conjugate groups comprise a cleavable moiety(e.g., a cleavable bond or cleavable nucleoside) and a carbohydratecluster portion, such as a GalNAc cluster portion. Such carbohydratecluster portion comprises: a targeting moiety and, optionally, aconjugate linker. In certain embodiments, the carbohydrate clusterportion is identified by the number and identity of the ligand. Forexample, in certain embodiments, the carbohydrate cluster portioncomprises 3 GalNAc groups and is designated “GalNAc₃”. In certainembodiments, the carbohydrate cluster portion comprises 4 GalNAc groupsand is designated “GalNAc₄”. Specific carbohydrate cluster portions(having specific tether, branching and conjugate linker groups) aredescribed herein and designated by Roman numeral followed by subscript“a”. Accordingly “GalNac3-1_(a)” refers to a specific carbohydratecluster portion of a conjugate group having 3 GalNac groups andspecifically identified tether, branching and linking groups. Suchcarbohydrate cluster fragment is attached to an oligomeric compound viaa cleavable moiety, such as a cleavable bond or cleavable nucleoside.

As used herein, “cleavable moiety” means a bond or group that is capableof being split under physiological conditions. In certain embodiments, acleavable moiety is cleaved inside a cell or sub-cellular compartments,such as a lysosome. In certain embodiments, a cleavable moiety iscleaved by endogenous enzymes, such as nucleases. In certainembodiments, a cleavable moiety comprises a group of atoms having one,two, three, four, or more than four cleavable bonds.

As used herein, “cleavable bond” means any chemical bond capable ofbeing split. In certain embodiments, a cleavable bond is selected fromamong: an amide, a polyamide, an ester, an ether, one or both esters ofa phosphodiester, a phosphate ester, a carbamate, a di-sulfide, or apeptide.

As used herein, “carbohydrate cluster” means a compound having one ormore carbohydrate residues attached to a scaffold or linker group. (see,e.g., Maier et al., “Synthesis of Antisense Oligonucleotides Conjugatedto a Multivalent Carbohydrate Cluster for Cellular Targeting,”Bioconjugate Chemistry, 2003, (14): 18-29, which is incorporated hereinby reference in its entirety, or Rensen et al., “Design and Synthesis ofNovel N-Acetylgalactosamine-Terminated Glycolipids for Targeting ofLipoproteins to the Hepatic Asiaglycoprotein Receptor,” J. Med. Chem.2004, (47): 5798-5808, for examples of carbohydrate conjugate clusters).

As used herein, “modified carbohydrate” means any carbohydrate havingone or more chemical modifications relative to naturally occurringcarbohydrates.

As used herein, “carbohydrate derivative” means any compound which maybe synthesized using a carbohydrate as a starting material orintermediate.

As used herein, “carbohydrate” means a naturally occurring carbohydrate,a modified carbohydrate, or a carbohydrate derivative.

As used herein “protecting group” means any compound or protecting groupknown to those having skill in the art. Non-limiting examples ofprotecting groups may be found in “Protective Groups in OrganicChemistry”, T. W. Greene, P. G. M. Wuts, ISBN 0-471-62301-6, John Wiley& Sons, Inc, New York, which is incorporated herein by reference in itsentirety.

As used herein, “single-stranded” means an oligomeric compound that isnot hybridized to its complement and which lacks sufficientself-complementarity to form a stable self-duplex.

As used herein, “double stranded” means a pair of oligomeric compoundsthat are hybridized to one another or a single self-complementaryoligomeric compound that forms a hairpin structure. In certainembodiments, a double-stranded oligomeric compound comprises a first anda second oligomeric compound.

As used herein, “antisense compound” means a compound comprising orconsisting of an oligonucleotide at least a portion of which iscomplementary to a target nucleic acid to which it is capable ofhybridizing, resulting in at least one antisense activity.

As used herein, “antisense activity” means any detectable and/ormeasurable change attributable to the hybridization of an antisensecompound to its target nucleic acid. In certain embodiments, antisenseactivity includes modulation of the amount or activity of a targetnucleic acid transcript (e.g. mRNA). In certain embodiments, antisenseactivity includes modulation of the splicing of pre-mRNA.

As used herein, “RNase H based antisense compound” means an antisensecompound wherein at least some of the antisense activity of theantisense compound is attributable to hybridization of the antisensecompound to a target nucleic acid and subsequent cleavage of the targetnucleic acid by RNase H.

As used herein, “RISC based antisense compound” means an antisensecompound wherein at least some of the antisense activity of theantisense compound is attributable to the RNA Induced Silencing Complex(RISC).

As used herein, “detecting” or “measuring” means that a test or assayfor detecting or measuring is performed. Such detection and/or measuringmay result in a value of zero. Thus, if a test for detection ormeasuring results in a finding of no activity (activity of zero), thestep of detecting or measuring the activity has nevertheless beenperformed.

As used herein, “detectable and/or measurable activity” means astatistically significant activity that is not zero.

As used herein, “essentially unchanged” means little or no change in aparticular parameter, particularly relative to another parameter whichchanges much more. In certain embodiments, a parameter is essentiallyunchanged when it changes less than 5%. In certain embodiments, aparameter is essentially unchanged if it changes less than two-foldwhile another parameter changes at least ten-fold. For example, incertain embodiments, an antisense activity is a change in the amount ofa target nucleic acid. In certain such embodiments, the amount of anon-target nucleic acid is essentially unchanged if it changes much lessthan the target nucleic acid does, but the change need not be zero.

As used herein, “expression” means the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, post-transcriptional modification (e.g., splicing,polyadenlyation, addition of 5′-cap), and translation.

As used herein, “target nucleic acid” means a nucleic acid molecule towhich an antisense compound is intended to hybridize to result in adesired antisense activity. Antisense oligonucleotides have sufficientcomplementarity to their target nucleic acids to allow hybridizationunder physiological conditions.

As used herein, “nucleobase complementarity” or “complementarity” whenin reference to nucleobases means a nucleobase that is capable of basepairing with another nucleobase. For example, in DNA, adenine (A) iscomplementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase means a nucleobase of an antisense compound that is capableof base pairing with a nucleobase of its target nucleic acid. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to becomplementary at that nucleobase pair. Nucleobases comprising certainmodifications may maintain the ability to pair with a counterpartnucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases means apair of nucleobases that do not form hydrogen bonds with one another.

As used herein, “complementary” in reference to oligomeric compounds(e.g., linked nucleosides, oligonucleotides, or nucleic acids) means thecapacity of such oligomeric compounds or regions thereof to hybridize toanother oligomeric compound or region thereof through nucleobasecomplementarity. Complementary oligomeric compounds need not havenucleobase complementarity at each nucleoside. Rather, some mismatchesare tolerated. In certain embodiments, complementary oligomericcompounds or regions are complementary at 70% of the nucleobases (70%complementary). In certain embodiments, complementary oligomericcompounds or regions are 80% complementary. In certain embodiments,complementary oligomeric compounds or regions are 90% complementary. Incertain embodiments, complementary oligomeric compounds or regions are95% complementary. In certain embodiments, complementary oligomericcompounds or regions are 100% complementary.

As used herein, “mismatch” means a nucleobase of a first oligomericcompound that is not capable of pairing with a nucleobase at acorresponding position of a second oligomeric compound, when the firstand second oligomeric compound are aligned. Either or both of the firstand second oligomeric compounds may be oligonucleotides.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleobases.

As used herein, “specifically hybridizes” means the ability of anoligomeric compound to hybridize to one nucleic acid site with greateraffinity than it hybridizes to another nucleic acid site.

As used herein, “fully complementary” in reference to an oligonucleotideor portion thereof means that each nucleobase of the oligonucleotide orportion thereof is capable of pairing with a nucleobase of acomplementary nucleic acid or contiguous portion thereof. Thus, a fullycomplementary region comprises no mismatches or unhybridized nucleobasesin either strand.

As used herein, “percent complementarity” means the percentage ofnucleobases of an oligomeric compound that are complementary to anequal-length portion of a target nucleic acid. Percent complementarityis calculated by dividing the number of nucleobases of the oligomericcompound that are complementary to nucleobases at correspondingpositions in the target nucleic acid by the total length of theoligomeric compound.

As used herein, “percent identity” means the number of nucleobases in afirst nucleic acid that are the same type (independent of chemicalmodification) as nucleobases at corresponding positions in a secondnucleic acid, divided by the total number of nucleobases in the firstnucleic acid.

As used herein, “modulation” means a change of amount or quality of amolecule, function, or activity when compared to the amount or qualityof a molecule, function, or activity prior to modulation. For example,modulation includes the change, either an increase (stimulation orinduction) or a decrease (inhibition or reduction) in gene expression.As a further example, modulation of expression can include a change insplice site selection of pre-mRNA processing, resulting in a change inthe absolute or relative amount of a particular splice-variant comparedto the amount in the absence of modulation.

As used herein, “chemical motif” means a pattern of chemicalmodifications in an oligonucleotide or a region thereof. Motifs may bedefined by modifications at certain nucleosides and/or at certainlinking groups of an oligonucleotide.

As used herein, “nucleoside motif” means a pattern of nucleosidemodifications in an oligonucleotide or a region thereof. The linkages ofsuch an oligonucleotide may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only nucleosides are intended to benucleoside motifs. Thus, in such instances, the linkages are notlimited.

As used herein, “sugar motif” means a pattern of sugar modifications inan oligonucleotide or a region thereof.

As used herein, “linkage motif” means a pattern of linkage modificationsin an oligonucleotide or region thereof. The nucleosides of such anoligonucleotide may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only linkages are intended to belinkage motifs. Thus, in such instances, the nucleosides are notlimited.

As used herein, “nucleobase modification motif” means a pattern ofmodifications to nucleobases along an oligonucleotide. Unless otherwiseindicated, a nucleobase modification motif is independent of thenucleobase sequence.

As used herein, “sequence motif” means a pattern of nucleobases arrangedalong an oligonucleotide or portion thereof. Unless otherwise indicated,a sequence motif is independent of chemical modifications and thus mayhave any combination of chemical modifications, including no chemicalmodifications.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” means the chemical modification of a nucleosideand includes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “differently modified” mean chemical modifications orchemical substituents that are different from one another, includingabsence of modifications. Thus, for example, a MOE nucleoside and anunmodified DNA nucleoside are “differently modified,” even though theDNA nucleoside is unmodified. Likewise, DNA and RNA are “differentlymodified,” even though both are naturally-occurring unmodifiednucleosides. Nucleosides that are the same but for comprising differentnucleobases are not differently modified. For example, a nucleosidecomprising a 2′-OMe modified sugar and an unmodified adenine nucleobaseand a nucleoside comprising a 2′-OMe modified sugar and an unmodifiedthymine nucleobase are not differently modified.

As used herein, “the same type of modifications” refers to modificationsthat are the same as one another, including absence of modifications.Thus, for example, two unmodified DNA nucleosides have “the same type ofmodification,” even though the DNA nucleoside is unmodified. Suchnucleosides having the same type modification may comprise differentnucleobases.

As used herein, “separate regions” means portions of an oligonucleotidewherein the chemical modifications or the motif of chemicalmodifications of any neighboring portions include at least onedifference to allow the separate regions to be distinguished from oneanother.

As used herein, “pharmaceutically acceptable carrier or diluent” meansany substance suitable for use in administering to an animal. In certainembodiments, a pharmaceutically acceptable carrier or diluent is sterilesaline. In certain embodiments, such sterile saline is pharmaceuticalgrade saline.

As used herein the term “metabolic disorder” means a disease orcondition principally characterized by dysregulation of metabolism—thecomplex set of chemical reactions associated with breakdown of food toproduce energy.

As used herein, the term “cardiovascular disorder” means a disease orcondition principally characterized by impaired function of the heart orblood vessels.

As used herein the term “mono or polycyclic ring system” is meant toinclude all ring systems selected from single or polycyclic radical ringsystems wherein the rings are fused or linked and is meant to beinclusive of single and mixed ring systems individually selected fromaliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl,heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl. Such monoand poly cyclic structures can contain rings that each have the samelevel of saturation or each, independently, have varying degrees ofsaturation including fully saturated, partially saturated or fullyunsaturated. Each ring can comprise ring atoms selected from C, N, O andS to give rise to heterocyclic rings as well as rings comprising only Cring atoms which can be present in a mixed motif such as for examplebenzimidazole wherein one ring has only carbon ring atoms and the fusedring has two nitrogen atoms. The mono or polycyclic ring system can befurther substituted with substituent groups such as for examplephthalimide which has two ═O groups attached to one of the rings. Monoor polycyclic ring systems can be attached to parent molecules usingvarious strategies such as directly through a ring atom, fused throughmultiple ring atoms, through a substituent group or through abifunctional linking moiety.

As used herein, “prodrug” means an inactive or less active form of acompound which, when administered to a subject, is metabolized to formthe active, or more active, compound (e.g., drug).

As used herein, “substituent” and “substituent group,” means an atom orgroup that replaces the atom or group of a named parent compound. Forexample a substituent of a modified nucleoside is any atom or group thatdiffers from the atom or group found in a naturally occurring nucleoside(e.g., a modified 2′-substituent is any atom or group at the 2′-positionof a nucleoside other than H or OH). Substituent groups can be protectedor unprotected. In certain embodiments, compounds of the presentdisclosure have substituents at one or at more than one position of theparent compound. Substituents may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemicalfunctional group means an atom or group of atoms that differs from theatom or a group of atoms normally present in the named functional group.In certain embodiments, a substituent replaces a hydrogen atom of thefunctional group (e.g., in certain embodiments, the substituent of asubstituted methyl group is an atom or group other than hydrogen whichreplaces one of the hydrogen atoms of an unsubstituted methyl group).Unless otherwise indicated, groups amenable for use as substituentsinclude without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl,acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups,alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl,heterocyclic radical, heteroaryl, heteroarylalkyl, amino(—N(R_(bb))(R_(cc))), imino (═NR_(bb)), amido (—C(O)N—(R_(bb))(R_(cc))or —N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro (—NO₂), cyano (—CN),carbamido (—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido(—N(R_(bb))C(O)N(R_(bb))(R_(cc))), thioureido(—N(R_(bb))C(S)N(R_(bb))(R_(cc))), guanidinyl(—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl(—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) andsulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S(O)₂R_(bb)). Whereineach R_(aa), R_(bb) and R_(cc) is, independently, H, an optionallylinked chemical functional group or a further substituent group with apreferred list including without limitation, alkyl, alkenyl, alkynyl,aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic,heterocyclic and heteroarylalkyl. Selected substituents within thecompounds described herein are present to a recursive degree.

As used herein, “alkyl,” as used herein, means a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include without limitation, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbonchain radical containing up to twenty four carbon atoms and having atleast one carbon-carbon double bond. Examples of alkenyl groups includewithout limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl,dienes such as 1,3-butadiene and the like. Alkenyl groups typicallyinclude from 2 to about 24 carbon atoms, more typically from 2 to about12 carbon atoms with from 2 to about 6 carbon atoms being morepreferred. Alkenyl groups as used herein may optionally include one ormore further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms and having at leastone carbon-carbon triple bond. Examples of alkynyl groups include,without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like.Alkynyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkynyl groups as used herein may optionallyinclude one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxylgroup from an organic acid and has the general Formula —C(O)—X where Xis typically aliphatic, alicyclic or aromatic. Examples includealiphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromaticsulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ringis aliphatic. The ring system can comprise one or more rings wherein atleast one ring is aliphatic. Preferred alicyclics include rings havingfrom about 5 to about 9 carbon atoms in the ring. Alicyclic as usedherein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms wherein the saturationbetween any two carbon atoms is a single, double or triple bond. Analiphatic group preferably contains from 1 to about 24 carbon atoms,more typically from 1 to about 12 carbon atoms with from 1 to about 6carbon atoms being more preferred. The straight or branched chain of analiphatic group may be interrupted with one or more heteroatoms thatinclude nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groupsinterrupted by heteroatoms include without limitation, polyalkoxys, suchas polyalkylene glycols, polyamines, and polyimines. Aliphatic groups asused herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl groupand an oxygen atom wherein the oxygen atom is used to attach the alkoxygroup to a parent molecule. Examples of alkoxy groups include withoutlimitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy,tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groupsas used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂ alkylradical. The alkyl portion of the radical forms a covalent bond with aparent molecule. The amino group can be located at any position and theaminoalkyl group can be substituted with a further substituent group atthe alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that iscovalently linked to a C₁-C₁₂ alkyl radical. The alkyl radical portionof the resulting aralkyl (or arylalkyl) group forms a covalent bond witha parent molecule. Examples include without limitation, benzyl,phenethyl and the like. Aralkyl groups as used herein may optionallyinclude further substituent groups attached to the alkyl, the aryl orboth groups that form the radical group.

As used herein, “aryl” and “aromatic” mean a mono- or polycycliccarbocyclic ring system radicals having one or more aromatic rings.Examples of aryl groups include without limitation, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ringsystems have from about 5 to about 20 carbon atoms in one or more rings.Aryl groups as used herein may optionally include further substituentgroups.

As used herein, “halo” and “halogen,” mean an atom selected fromfluorine, chlorine, bromine and iodine.

As used herein, “heteroaryl,” and “heteroaromatic,” mean a radicalcomprising a mono- or poly-cyclic aromatic ring, ring system or fusedring system wherein at least one of the rings is aromatic and includesone or more heteroatoms. Heteroaryl is also meant to include fused ringsystems including systems where one or more of the fused rings containno heteroatoms. Heteroaryl groups typically include one ring atomselected from sulfur, nitrogen or oxygen. Examples of heteroaryl groupsinclude without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroarylradicals can be attached to a parent molecule directly or through alinking moiety such as an aliphatic group or hetero atom. Heteroarylgroups as used herein may optionally include further substituent groups.

As used herein, “conjugate compound” means any atoms, group of atoms, orgroup of linked atoms suitable for use as a conjugate group. In certainembodiments, conjugate compounds may possess or impart one or moreproperties, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, unless otherwise indicated or modified, the term“double-stranded” refers to two separate oligomeric compounds that arehybridized to one another. Such double stranded compounds may have oneor more or non-hybridizing nucleosides at one or both ends of one orboth strands (overhangs) and/or one or more internal non-hybridizingnucleosides (mismatches) provided there is sufficient complementarity tomaintain hybridization under physiologically relevant conditions.

As used herein, “5′ target site” refers to the nucleotide of a targetnucleic acid which is complementary to the 5′-most nucleotide of aparticular antisense compound.

As used herein, “About” means within ±10% of a value. For example, if itis stated, “a marker may be increased by about 50%”, it is implied thatthe marker may be increased between 45%-55%.

As used herein, “administered concomitantly” refers to theco-administration of two agents in any manner in which thepharmacological effects of both are manifest in the patient at the sametime. Concomitant administration does not require that both agents beadministered in a single pharmaceutical composition, in the same dosageform, or by the same route of administration. The effects of both agentsneed not manifest themselves at the same time. The effects need only beoverlapping for a period of time and need not be coextensive.

As used herein, “administering” or “administration” means providing apharmaceutical agent to an individual, and includes, but is not limitedto, administering by a medical professional and self-administering.Administration of a pharmaceutical agent to an individual can becontinuous, chronic, short or intermittent. Administration canparenteral or non-parenteral.

As used herein, “agent” means an active substance that can provide atherapeutic benefit when administered to an animal. “First agent” meansa therapeutic compound of the invention. For example, a first agent canbe an antisense oligonucleotide targeting apo(a). “Second agent” means asecond therapeutic compound of the invention (e.g. a second antisenseoligonucleotide targeting apo(a)) and/or a non-apo(a) therapeuticcompound.

As used herein, “amelioration” or “ameliorate” or “ameliorating” refersto a lessening of at least one indicator, sign, or symptom of anassociated disease, disorder, or condition. The severity of indicatorscan be determined by subjective or objective measures, which are knownto those skilled in the art.

As used herein, “animal” refers to a human or non-human animal,including, but not limited to, mice, rats, rabbits, dogs, cats, pigs,and non-human primates, including, but not limited to, monkeys andchimpanzees.

As used herein, “apo(a)” means any nucleic acid or protein sequenceencoding apo(a). For example, in certain embodiments, apo(a) includes aDNA sequence encoding apo(a), a RNA sequence transcribed from DNAencoding apo(a) (including genomic DNA comprising introns and exons), amRNA sequence encoding apo(a), or a peptide sequence encoding apo(a).

As used herein, “apo(a) nucleic acid” means any nucleic acid encodingapo(a). For example, in certain embodiments, an apo(a) nucleic acidincludes a DNA sequence encoding apo(a), a RNA sequence transcribed fromDNA encoding apo(a) (including genomic DNA comprising introns andexons), and a mRNA sequence encoding apo(a).

As used herein, “apo(a) mRNA” means a mRNA encoding an apo(a) protein.

As used herein, “apo(a) protein” means any protein sequence encodingApo(a).

As used herein, “apo(a) specific inhibitor” refers to any agent capableof specifically inhibiting the expression of an apo(a) nucleic acidand/or apo(a) protein. For example, apo(a) specific inhibitors includenucleic acids (including antisense compounds), peptides, antibodies,small molecules, and other agents capable of inhibiting the expressionof apo(a) nucleic acid and/or apo(a) protein. In certain embodiments, byspecifically modulating apo(a) nucleic acid expression and/or apo(a)protein expression, apo(a) specific inhibitors can affect othercomponents of the lipid transport system including downstreamcomponents. Similarly, in certain embodiments, apo(a) specificinhibitors can affect other molecular processes in an animal.

As used herein, “atherosclerosis” means a hardening of the arteriesaffecting large and medium-sized arteries and is characterized by thepresence of fatty deposits. The fatty deposits are called “atheromas” or“plaques,” which consist mainly of cholesterol and other fats, calciumand scar tissue, and damage the lining of arteries.

As used herein, “coronary heart disease (CHD)” means a narrowing of thesmall blood vessels that supply blood and oxygen to the heart, which isoften a result of atherosclerosis.

As used herein, “diabetes mellitus” or “diabetes” is a syndromecharacterized by disordered metabolism and abnormally high blood sugar(hyperglycemia) resulting from insufficient levels of insulin or reducedinsulin sensitivity. The characteristic symptoms are excessive urineproduction (polyuria) due to high blood glucose levels, excessive thirstand increased fluid intake (polydipsia) attempting to compensate forincreased urination, blurred vision due to high blood glucose effects onthe eye's optics, unexplained weight loss, and lethargy.

As used herein, “diabetic dyslipidemia” or “type 2 diabetes withdyslipidemia” means a condition characterized by Type 2 diabetes,reduced HDL-C, elevated triglycerides (TG), and elevated small, denseLDL particles.

As used herein, “diluent” means an ingredient in a composition thatlacks pharmacological activity, but is pharmaceutically necessary ordesirable. For example, the diluent in an injected composition can be aliquid, e.g. saline solution.

As used herein, “dyslipidemia” refers to a disorder of lipid and/orlipoprotein metabolism, including lipid and/or lipoproteinoverproduction or deficiency. Dyslipidemias can be manifested byelevation of lipids such as chylomicron, cholesterol and triglyceridesas well as lipoproteins such as low-density lipoprotein (LDL)cholesterol.

As used herein, “dosage unit” means a form in which a pharmaceuticalagent is provided, e.g. pill, tablet, or other dosage unit known in theart. In certain embodiments, a dosage unit is a vial containinglyophilized antisense oligonucleotide. In certain embodiments, a dosageunit is a vial containing reconstituted antisense oligonucleotide.

As used herein, “dose” means a specified quantity of a pharmaceuticalagent provided in a single administration, or in a specified timeperiod. In certain embodiments, a dose can be administered in one, two,or more boluses, tablets, or injections. For example, in certainembodiments where subcutaneous administration is desired, the desireddose requires a volume not easily accommodated by a single injection,therefore, two or more injections can be used to achieve the desireddose. In certain embodiments, the pharmaceutical agent is administeredby infusion over an extended period of time or continuously. Doses canbe stated as the amount of pharmaceutical agent per hour, day, week, ormonth. Doses can also be stated as mg/kg or g/kg.

As used herein, “effective amount” or “therapeutically effective amount”means the amount of active pharmaceutical agent sufficient to effectuatea desired physiological outcome in an individual in need of the agent.The effective amount can vary among individuals depending on the healthand physical condition of the individual to be treated, the taxonomicgroup of the individuals to be treated, the formulation of thecomposition, assessment of the individual's medical condition, and otherrelevant factors.

As used herein, “fully complementary” or “100% complementary” means eachnucleobase of a nucleobase sequence of a first nucleic acid has acomplementary nucleobase in a second nucleobase sequence of a secondnucleic acid. In certain embodiments, a first nucleic acid is anantisense compound and a second nucleic acid is a target nucleic acid.

As used herein, “glucose” is a monosaccharide used by cells as a sourceof energy and inflammatory intermediate. “Plasma glucose” refers toglucose present in the plasma.

As used herein, “high density lipoprotein-C” or “HDL-C” meanscholesterol associated with high density lipoprotein particles.Concentration of HDL-C in serum (or plasma) is typically quantified inmg/dL or nmol/L. “Serum HDL-C” and “plasma HDL-C” mean HDL-C in serumand plasma, respectively.

As used herein, “HMG-CoA reductase inhibitor” means an agent that actsthrough the inhibition of the enzyme HMG-CoA reductase, such asatorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, andsimvastatin.

As used herein, “hypercholesterolemia” means a condition characterizedby elevated cholesterol or circulating (plasma) cholesterol,LDL-cholesterol and VLDL-cholesterol, as per the guidelines of theExpert Panel Report of the National Cholesterol Educational Program(NCEP) of Detection, Evaluation of Treatment of high cholesterol inadults (see, Arch. Int. Med. (1988) 148, 36-39).

As used herein, “hyperlipidemia” or “hyperlipemia” is a conditioncharacterized by elevated serum lipids or circulating (plasma) lipids.This condition manifests an abnormally high concentration of fats. Thelipid fractions in the circulating blood are cholesterol, low densitylipoproteins, very low density lipoproteins, chylomicrons andtriglycerides. The Fredrickson classification of hyperlipidemias isbased on the pattern of TG and cholesterol-rich lipoprotein particles,as measured by electrophoresis or ultracentrifugation and is commonlyused to characterize primary causes of hyperlipidemias such ashypertriglyceridemia (Fredrickson and Lee, Circulation, 1965,31:321-327; Fredrickson et al., New Eng J Med, 1967, 276 (1): 34-42).

As used herein, “hypertriglyceridemia” means a condition characterizedby elevated triglyceride levels. Its etiology includes primary (i.e.genetic causes) and secondary (other underlying causes such as diabetes,metabolic syndrome/insulin resistance, obesity, physical inactivity,cigarette smoking, excess alcohol and a diet very high in carbohydrates)factors or, most often, a combination of both (Yuan et al. CMAJ, 2007,176:1113-1120).

As used herein, “identifying” or “selecting an animal with metabolic orcardiovascular disease” means identifying or selecting a subject proneto or having been diagnosed with a metabolic disease, a cardiovasculardisease, or a metabolic syndrome; or, identifying or selecting a subjecthaving any symptom of a metabolic disease, cardiovascular disease, ormetabolic syndrome including, but not limited to, hypercholesterolemia,hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertensionincreased insulin resistance, decreased insulin sensitivity, abovenormal body weight, and/or above normal body fat content or anycombination thereof. Such identification can be accomplished by anymethod, including but not limited to, standard clinical tests orassessments, such as measuring serum or circulating (plasma)cholesterol, measuring serum or circulating (plasma) blood-glucose,measuring serum or circulating (plasma) triglycerides, measuringblood-pressure, measuring body fat content, measuring body weight, andthe like.

As used herein, “improved cardiovascular outcome” means a reduction inthe occurrence of adverse cardiovascular events, or the risk thereof.Examples of adverse cardiovascular events include, without limitation,death, reinfarction, stroke, cardiogenic shock, pulmonary edema, cardiacarrest, and atrial dysrhythmia.

As used herein, “immediately adjacent” means there are no interveningelements between the immediately adjacent elements, for example, betweenregions, segments, nucleotides and/or nucleosides.

As used herein, “increasing HDL” or “raising HDL” means increasing thelevel of HDL in an animal after administration of at least one compoundof the invention, compared to the HDL level in an animal notadministered any compound.

As used herein, “individual” or “subject” or “animal” means a human ornon-human animal selected for treatment or therapy.

As used herein, “individual in need thereof” refers to a human ornon-human animal selected for treatment or therapy that is in need ofsuch treatment or therapy.

As used herein, “induce”, “inhibit”, “potentiate”, “elevate”,“increase”, “decrease”, “reduce” or the like denote quantitativedifferences between two states. For example, “an amount effective toinhibit the activity or expression of apo(a)” means that the level ofactivity or expression of apo(a) in a treated sample will differ fromthe level of apo(a) activity or expression in an untreated sample. Suchterms are applied to, for example, levels of expression, and levels ofactivity.

As used herein, “inflammatory condition” refers to a disease, diseasestate, syndrome, or other condition resulting in inflammation. Forexample, rheumatoid arthritis and liver fibrosis are inflammatoryconditions. Other examples of inflammatory conditions include sepsis,myocardial ischemia/reperfusion injury, adult respiratory distresssyndrome, nephritis, graft rejection, inflammatory bowel disease,multiple sclerosis, arteriosclerosis, atherosclerosis and vasculitis.

As used herein, “inhibiting the expression or activity” refers to areduction or blockade of the expression or activity of a RNA or proteinand does not necessarily indicate a total elimination of expression oractivity.

As used herein, “insulin resistance” is defined as the condition inwhich normal amounts of insulin are inadequate to produce a normalinsulin response from fat, muscle and liver cells. Insulin resistance infat cells results in hydrolysis of stored triglycerides, which elevatesfree fatty acids in the blood plasma. Insulin resistance in musclereduces glucose uptake whereas insulin resistance in liver reducesglucose storage, with both effects serving to elevate blood glucose.High plasma levels of insulin and glucose due to insulin resistanceoften leads to metabolic syndrome and type 2 diabetes.

As used herein, “insulin sensitivity” is a measure of how effectively anindividual processes glucose. An individual having high insulinsensitivity effectively processes glucose whereas an individual with lowinsulin sensitivity does not effectively process glucose.

As used herein, “lipid-lowering” means a reduction in one or more lipids(e.g., LDL, VLDL) in a subject. “Lipid-raising” means an increase in alipid (e.g., HDL) in a subject. Lipid-lowering or lipid-raising canoccur with one or more doses over time.

As used herein, “lipid-lowering therapy” or “lipid lowering agent” meansa therapeutic regimen provided to a subject to reduce one or more lipidsin a subject. In certain embodiments, a lipid-lowering therapy isprovided to reduce one or more of apo(a), CETP, apoB, total cholesterol,LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small dense LDLparticles, and Lp(a) in a subject. Examples of lipid-lowering therapyinclude, but are not limited to, apoB inhibitors, statins, fibrates andMTP inhibitors.

As used herein, “lipoprotein”, such as VLDL, LDL and HDL, refers to agroup of proteins found in the serum, plasma and lymph and are importantfor lipid transport. The chemical composition of each lipoproteindiffers, for example, in that the HDL has a higher proportion of proteinversus lipid, whereas the VLDL has a lower proportion of protein versuslipid.

As used herein, “Lp(a)” comprises apo(a) and a LDL like particlecontaining apoB. The apo(a) is linked to the apoB by a disulfide bond.

As used herein, “low density lipoprotein-cholesterol (LDL-C)” meanscholesterol carried in low density lipoprotein particles. Concentrationof LDL-C in serum (or plasma) is typically quantified in mg/dL ornmol/L. “Serum LDL-C” and “plasma LDL-C” mean LDL-C in the serum andplasma, respectively.

As used herein, “major risk factors” refers to factors that contributeto a high risk for a particular disease or condition. In certainembodiments, major risk factors for coronary heart disease include,without limitation, cigarette smoking, hypertension, high LDL, lowHDL-C, family history of coronary heart disease, age, and other factorsdisclosed herein.

As used herein, “metabolic disorder” or “metabolic disease” refers to acondition characterized by an alteration or disturbance in metabolicfunction. “Metabolic” and “metabolism” are terms well known in the artand generally include the whole range of biochemical processes thatoccur within a living organism. Metabolic disorders include, but are notlimited to, hyperglycemia, prediabetes, diabetes (type 1 and type 2),obesity, insulin resistance, metabolic syndrome and dyslipidemia due totype 2 diabetes.

As used herein, “metabolic syndrome” means a condition characterized bya clustering of lipid and non-lipid cardiovascular risk factors ofmetabolic origin. In certain embodiments, metabolic syndrome isidentified by the presence of any 3 of the following factors: waistcircumference of greater than 102 cm in men or greater than 88 cm inwomen; serum triglyceride of at least 150 mg/dL; HDL-C less than 40mg/dL in men or less than 50 mg/dL in women; blood pressure of at least130/85 mmHg; and fasting glucose of at least 110 mg/dL. Thesedeterminants can be readily measured in clinical practice (JAMA, 2001,285: 2486-2497).

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes subcutaneousadministration, intravenous administration, intramuscularadministration, intraarterial administration, intraperitonealadministration, or intracranial administration, e.g. intrathecal orintracerebroventricular administration. Administration can becontinuous, chronic, short or intermittent.

As used herein, “peptide” means a molecule formed by linking at leasttwo amino acids by amide bonds. Peptide refers to polypeptides andproteins.

As used herein, “pharmaceutical agent” means a substance that provides atherapeutic benefit when administered to an individual. For example, incertain embodiments, an antisense oligonucleotide targeted to apo(a) isa pharmaceutical agent.

As used herein, “pharmaceutical composition” or “composition” means amixture of substances suitable for administering to an individual. Forexample, a pharmaceutical composition can comprise one or more activeagents and a pharmaceutical carrier e.g., a sterile aqueous solution.

As used herein, “pharmaceutically acceptable derivative” encompassesderivatives of the compounds described herein such as solvates,hydrates, esters, prodrugs, polymorphs, isomers, isotopically labelledvariants, pharmaceutically acceptable salts and other derivatives knownin the art.

As used herein, “pharmaceutically acceptable salts” meansphysiologically and pharmaceutically acceptable salts of antisensecompounds, i.e., salts that retain the desired biological activity ofthe parent compound and do not impart undesired toxicological effectsthereto. The term “pharmaceutically acceptable salt” or “salt” includesa salt prepared from pharmaceutically acceptable non-toxic acids orbases, including inorganic or organic acids and bases. “Pharmaceuticallyacceptable salts” of the compounds described herein may be prepared bymethods well-known in the art. For a review of pharmaceuticallyacceptable salts, see Stahl and Wermuth, Handbook of PharmaceuticalSalts: Properties, Selection and Use (Wiley-VCH, Weinheim, Germany,2002). Sodium salts of antisense oligonucleotides are useful and arewell accepted for therapeutic administration to humans. Accordingly, inone embodiment the compounds described herein are in the form of asodium salt.

As used herein, “portion” means a defined number of contiguous (i.e.linked) nucleobases of a nucleic acid. In certain embodiments, a portionis a defined number of contiguous nucleobases of a target nucleic acid.In certain embodiments, a portion is a defined number of contiguousnucleobases of an antisense compound.

As used herein, “prevent” or “preventing” refers to delaying orforestalling the onset or development of a disease, disorder, orcondition for a period of time from minutes to indefinitely. Preventalso means reducing risk of developing a disease, disorder, orcondition.

As used herein, “raise” means to increase in amount. For example, toraise plasma HDL levels means to increase the amount of HDL in theplasma.

As used herein, “reduce” means to bring down to a smaller extent, size,amount, or number. For example, to reduce plasma triglyceride levelsmeans to bring down the amount of triglyceride in the plasma.

As used herein, “region” or “target region” is defined as a portion ofthe target nucleic acid having at least one identifiable structure,function, or characteristic. For example, a target region may encompassa 3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, acoding region, a translation initiation region, translation terminationregion, or other defined nucleic acid region. The structurally definedregions for apo(a) can be obtained by accession number from sequencedatabases such as NCBI and such information is incorporated herein byreference. In certain embodiments, a target region may encompass thesequence from a 5′ target site of one target segment within the targetregion to a 3′ target site of another target segment within the targetregion.

As used herein, “second agent” or “second therapeutic agent” means anagent that can be used in combination with a “first agent”. A secondtherapeutic agent can include, but is not limited to, antisenseoligonucleotides targeting apo(a) or apoB. A second agent can alsoinclude anti-apo(a) antibodies, apo(a) peptide inhibitors, cholesterollowering agents, lipid lowering agents, glucose lowering agents andanti-inflammatory agents.

As used herein, “segments” are defined as smaller, sub-portions ofregions within a nucleic acid. For example, a “target segment” means thesequence of nucleotides of a target nucleic acid to which one or moreantisense compounds is targeted. “5′ target site” refers to the 5′-mostnucleotide of a target segment. “3′ target site” refers to the 3′-mostnucleotide of a target segment. Alternatively, a “start site” can referto the 5′-most nucleotide of a target segment and a “stop site” refersto the 3′-most nucleotide of a target segment. A target segment can alsobegin at the “start site” of one sequence and end at the “stop site” ofanother sequence.

As used herein, “statin” means an agent that inhibits the activity ofHMG-CoA reductase.

As used herein, “subcutaneous administration” means administration justbelow the skin.

As used herein, “subject” means a human or non-human animal selected fortreatment or therapy.

As used herein, “symptom of cardiovascular disease or disorder” means aphenomenon that arises from and accompanies the cardiovascular diseaseor disorder and serves as an indication of it. For example, angina;chest pain; shortness of breath; palpitations; weakness; dizziness;nausea; sweating; tachycardia; bradycardia; arrhythmia; atrialfibrillation; swelling in the lower extremities; cyanosis; fatigue;fainting; numbness of the face; numbness of the limbs; claudication orcramping of muscles; bloating of the abdomen; or fever are symptoms ofcardiovascular disease or disorder.

As used herein, “targeting” or “targeted” means the process of designand selection of an antisense compound that will specifically hybridizeto a target nucleic acid and induce a desired effect.

As used herein, “therapeutically effective amount” means an amount of apharmaceutical agent that provides a therapeutic benefit to anindividual.

As used herein, “therapeutic lifestyle change” means dietary andlifestyle changes intended to lower fat/adipose tissue mass and/orcholesterol. Such change can reduce the risk of developing heartdisease, and may includes recommendations for dietary intake of totaldaily calories, total fat, saturated fat, polyunsaturated fat,monounsaturated fat, carbohydrate, protein, cholesterol, insolublefiber, as well as recommendations for physical activity.

As used herein, “treat” or “treating” refers to administering a compounddescribed herein to effect an alteration or improvement of a disease,disorder, or condition.

As used herein, “triglyceride” or “TG” means a lipid or neutral fatconsisting of glycerol combined with three fatty acid molecules.

As used herein, “type 2 diabetes,” (also known as “type 2 diabetesmellitus”, “diabetes mellitus, type 2”, “non-insulin-dependentdiabetes”, “NIDDM”, “obesity related diabetes”, or “adult-onsetdiabetes”) is a metabolic disorder that is primarily characterized byinsulin resistance, relative insulin deficiency, and hyperglycemia.

Certain Embodiments

In certain embodiments, a compound comprises a siRNA or antisenseoligonucleotide targeted to apolipoprotein(a) (apo(a)) known in the artand a conjugate group described herein. Examples of antisenseoligonucleotides targeted to apo(a) suitable for conjugation include butare not limited to those disclosed in WO 2013/177468; U.S. Pat. Nos.8,673,632; 7,259,150; and US Patent Application Publication No. US2004/0242516; which are incorporated by reference in their entiretiesherein. In certain embodiments, a compound comprises an antisenseoligonucleotide having a nucleobase sequence of any of SEQ ID NOs12-130, 133, 134 disclosed in WO 2013/177468 and a conjugate groupdescribed herein. In certain embodiments, a compound comprises anantisense oligonucleotide having a nucleobase sequence of any of SEQ IDNOs 11-45 and 85-96 disclosed in U.S. Pat. No. 8,673,632 and a conjugategroup described herein. In certain embodiments, a compound comprises anantisense oligonucleotide having a nucleobase sequence of any of SEQ IDNOs 11-45 disclosed in U.S. Pat. No. 7,259,150 and a conjugate groupdescribed herein. In certain embodiments, a compound comprises anantisense oligonucleotide having a nucleobase sequence of any of SEQ IDNOs 7-41 disclosed in US Patent Application Publication No. US2004/0242516 and a conjugate group described herein. The nucleobasesequences of all of the aforementioned referenced SEQ ID NOs areincorporated by reference herein.

Certain embodiments provide a compounds and methods for decreasingapo(a) mRNA and protein expression. In certain embodiments, the compoundis an apo(a) specific inhibitor for treating, preventing, orameliorating an apo(a) associated disease. In certain embodiments, thecompound is an antisense oligonucleotide targeting apo(a). In certainembodiments, the compound is an antisense oligonucleotide targetingapo(a) and a conjugate group.

Certain embodiments provide a compounds and methods for decreasing Lp(a)levels. In certain embodiments, the compound is an apo(a) specificinhibitor for treating, preventing, or ameliorating an Lp(a) associateddisease. In certain embodiments, the compound is an antisenseoligonucleotide targeting apo(a). In certain embodiments, the compoundis an antisense oligonucleotide targeting apo(a) and a conjugate group.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides. Incertain embodiments, the modified oligonucleotide with the conjugategroup consists of 15 to 30, 18 to 24, 19 to 22, 13 to 25, 14 to 25, 15to 25 linked nucleosides. In certain embodiments, the modifiedoligonucleotide with the conjugate group comprises at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 28, at least 29 or30 linked nucleosides. In certain embodiments, the modifiedoligonucleotide with the conjugate group consists of 20 linkednucleosides.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide comprises at least 8, at least 9, at least 10,at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or 20 contiguousnucleobases complementary to an equal length portion of any of SEQ IDNOs: 1-4.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting an apo(a) segment and a conjugate group,wherein the modified oligonucleotide comprises at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, or 20contiguous nucleobases complementary to an equal length portion of anyof the target segments shown in, for example, Examples 114 and 117. Inthe tables, the “Start Site” refers to the 5′-most nucleotide of atarget segment and “Stop Site” refers to the 3′-most nucleotide of atarget segment. A target segment can range from the start site to thestop site of each sequence listed in the tables. Alternatively, thetarget segment can range from the start site of one sequence and end atthe stop site of another sequence. For example, as shown in Table 125, atarget segment can range from 3901-3920, the start site to the stop siteof SEQ ID NO: 58. In another example, as shown in Table 125, a targetsegment can range from 3900-3923, the start site of SEQ ID NO: 57 to thestop site of SEQ ID NO: 61.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein thenucleobase sequence of the modified oligonucleotide is at least 80%, atleast 85%, at least 90%, at least 95%, or 100% complementary to any ofSEQ ID NOs: 1-4. Certain embodiments provide a compound comprising amodified oligonucleotide targeting apo(a) and a conjugate group, whereinthe nucleobase sequence of the modified oligonucleotide is at least 80%,at least 85%, at least 90%, at least 95%, or 100% complementary to anyof the target segments shown in, for example, Examples 114 and 117.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andcomprises a nucleobase sequence comprising a portion of at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, or20 contiguous nucleobases complementary to an equal length portion ofnucleobases 3901 to 3920 of SEQ ID NO: 1, wherein the nucleobasesequence of the modified oligonucleotide is at least 80% complementaryto SEQ ID NO: 1.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andcomprises a nucleobase sequence comprising at least 8, at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, at least 27, at least 28, at least 29 or 30 contiguous nucleobasescomplementary to an equal length portion of nucleobases 3900 to 3923 ofSEQ ID NO: 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 80% complementary to SEQ ID NO: 1.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides and hasa nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or 20 contiguous nucleobasesof any of the nucleobase sequences of SEQ ID NOs: 12-130, 133, 134. Incertain embodiments, the modified oligonucleotide has a nucleobasesequence comprising at least 8 contiguous nucleobases of any one of thenucleobase sequences of SEQ ID NOs: 12-130, 133, 134. In certainembodiments, the compound consists of any one of SEQ ID NOs: 12-130,133, 134 and a conjugate group.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides and hasa nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or 20 contiguous nucleobasesof any of the nucleobase sequences of SEQ ID NOs: 12-20, 22-33, 35-44,47-50, 51, 53, 57-62, 65-66, 68, 70-79, 81, 85-86, 89-90, 92-94, 97,105-110, 103-104, 133-134. In certain embodiments, the compound consistsof any of the nucleobase sequences of SEQ ID NOs: 12-20, 22-33, 35-44,47-50, 51, 53, 57-62, 65-66, 68, 70-79, 81, 85-86, 89-90, 92-94, 97,105-110, 103-104, 133-134 and a conjugate group.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides and hasa nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or 20 contiguous nucleobasesof any of the nucleobase sequences of SEQ ID NOs: 12-19, 26-30, 32, 35,38-44, 46-47, 50, 57-58, 61, 64-66, 68, 72-74, 76-77, 92-94, 103-110. Incertain embodiments, the compound consists of any of the nucleobasesequences of SEQ ID NOs: 12-19, 26-30, 32, 35, 38-44, 46-47, 50, 57-58,61, 64-66, 68, 72-74, 76-77, 92-94, 103-110 and a conjugate group.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides and hasa nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or 20 contiguous nucleobasesof any of the nucleobase sequences of SEQ ID NOs: 111, 114-121, 123-129.In certain embodiments, the compound consists of any of the nucleobasesequences of SEQ ID NOs: 111, 114-121, 123-129 and a conjugate group.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides and hasa nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or 20 contiguous nucleobasesof any of the nucleobase sequences of SEQ ID NOs: 14, 17, 18, 26-28, 39,71, 106-107. In certain embodiments, the compound consists of any of thenucleobase sequences of SEQ ID NOs: 14, 17, 18, 26-28, 39, 71, 106-107and a conjugate group.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides and hasa nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or 20 contiguous nucleobasesof any of the nucleobase sequences of SEQ ID NOs: 14, 26-29, 39-40, 82.In certain embodiments, the compound consists of any of the nucleobasesequences of SEQ ID NOs: 14, 26-29, 39-40, 82 and a conjugate group.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides and hasa nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or 20 contiguous nucleobasesof any of the nucleobase sequences of SEQ ID NOs: 14, 16-18. In certainembodiments, the compound consists of any of the nucleobase sequences ofSEQ ID NOs: 14, 16-18 and a conjugate group.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides and hasa nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or 20 contiguous nucleobasesof any of the nucleobase sequences of SEQ ID NOs: 26-27, 107. In certainembodiments, the compound consists of any of the nucleobase sequences ofSEQ ID NOs: 26-27, 107 and a conjugate group.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides and hasa nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or 20 contiguous nucleobasesof any of the nucleobase sequences of SEQ ID NOs: 28-29, 39-40, 47. Incertain embodiments, the compound consists of any of the nucleobasesequences of SEQ ID NOs: 28-29, 39-40, 47 and a conjugate group.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides and hasa nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or 20 contiguous nucleobasesof any of the nucleobase sequences of SEQ ID NOs: 28, 93, 104, 134. Incertain embodiments, the compound consists of any of the nucleobasesequences of SEQ ID NOs: 28, 93, 104, 134 and a conjugate group.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides and hasa nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or 20 contiguous nucleobasesof the nucleobase sequence of SEQ ID NO: 58. In certain embodiments, themodified oligonucleotide with the conjugate group has a nucleobasesequence comprising at least 8 contiguous nucleobases of the nucleobasesequence of SEQ ID NO: 58. In certain embodiments, the compound consistsof SEQ ID NO: 58 and a conjugate group.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the modifiedoligonucleotide ISIS 494372 with a 5′-X, wherein X is a conjugate groupcomprising GalNAc. In certain embodiments, the antisense compoundconsists of the modified oligonucleotide ISIS 494372 with a 5′-X,wherein X is a conjugate group comprising GalNAc.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the conjugated modifiedoligonucleotide ISIS 681251. In certain embodiments, the antisensecompound consists of the conjugated modified oligonucleotide ISIS681251.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the conjugated modifiedoligonucleotide ISIS 681257. In certain embodiments, the antisensecompound consists of the conjugated modified oligonucleotide ISIS681257.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises a modified oligonucleotidewith the nucleobase sequence of SEQ ID NO: 58 with a 5′-GalNAc withvariability in the sugar mods of the wings. In certain embodiments, theantisense compound consists of a modified oligonucleotide with thenucleobase sequence of SEQ ID NO: 58 with a 5′-GalNAc with variabilityin the sugar mods of the wings.

Wherein either R¹ is —OCH₂CH₂OCH₃ (MOE) and R² is H; or R¹ and R²together form a bridge, wherein R′ is —O— and R² is —CH₂—, —CH(CH₃)—, or—CH₂CH₂—, and R¹ and R² are directly connected such that the resultingbridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

And for each pair of R³ and R⁴ on the same ring, independently for eachring: either R³ is selected from H and —OCH₂CH₂OCH₃ and R⁴ is H; or R³and together form a bridge, wherein R³ is —O—, and R⁴ is —CH₂—,—CH(CH₃)—, or —CH₂CH₂— and R³ and are directly connected such that theresulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

And R⁵ is selected from H and —CH₃;

And Z is selected from S⁻ and

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide is single-stranded.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein at leastone internucleoside linkage is a modified internucleoside linkage. Incertain embodiments, the modified internucleoside linkage is aphosphorothioate internucleoside linkage. In certain embodiments, atleast 1, at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9 or at least 10 internucleoside linkagesof said modified oligonucleotide are phosphorothioate internucleosidelinkages. In certain embodiments, each internucleoside linkage is aphosphorothioate internucleoside linkage. In certain embodiments, themodified oligonucleotide comprises at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9or at least 10 phosphodiester internucleoside linkages. In certainembodiments, each internucleoside linkage of the modifiedoligonucleotide is selected from a phosphodiester internucleosidelinkage and a phosphorothioate internucleoside linkage.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein at leastone nucleoside comprises a modified nucleobase. In certain embodiments,the modified nucleobase is a 5-methylcytosine.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide comprises at least one modified sugar. Incertain embodiments, the modified sugar is a bicyclic sugar. In certainembodiments, the modified sugar comprises a 2′-O-methoxyethyl, aconstrained ethyl, a 3′-fluoro-HNA or a 4′-(CH₂)_(n)—O-2′ bridge,wherein n is 1 or 2.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andcomprises: (a) a gap segment consisting of linked deoxynucleosides; (b)a 5′ wing segment consisting of linked nucleosides; (c) a 3′ wingsegment consisting of linked nucleosides; and wherein the gap segment ispositioned between the 5′ wing segment and the 3′ wing segment andwherein each nucleoside of each wing segment comprises a modified sugar.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 20 linked nucleosides andcomprises: (a) a gap segment consisting of ten linked deoxynucleosides;(b) a 5′ wing segment consisting of five linked nucleosides; (c) a 3′wing segment consisting of five linked nucleosides; and wherein the gapsegment is positioned between the 5′ wing segment and the 3′ wingsegment, wherein each nucleoside of each wing segment comprises a2′-O-methoxyethyl sugar, wherein at least one internucleoside linkage isa phosphorothioate linkage and wherein each cytosine residue is a5-methylcytosine.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 20 linked nucleosides and has anucleobase sequence comprising at least 8 contiguous nucleobases of anyof SEQ ID NOs: 12-130, 133, 134, wherein the modified oligonucleotidecomprises: (a) a gap segment consisting of ten linked deoxynucleosides;(b) a 5′ wing segment consisting of five linked nucleosides; (c) a 3′wing segment consisting of five linked nucleosides; and wherein the gapsegment is positioned between the 5′ wing segment and the 3′ wingsegment, wherein each nucleoside of each wing segment comprises a2′-O-methoxyethyl sugar, wherein at least one internucleoside linkage isa phosphorothioate linkage and wherein each cytosine residue is a5-methylcytosine.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting apo(a) and a conjugate group, wherein themodified oligonucleotide consists of 20 linked nucleosides and has anucleobase sequence comprising at least 8 contiguous nucleobases of SEQID NO: 58, wherein the modified oligonucleotide comprises: (a) a gapsegment consisting of ten linked deoxynucleosides; (b) a 5′ wing segmentconsisting of five linked nucleosides; (c) a 3′ wing segment consistingof five linked nucleosides; and wherein the gap segment is positionedbetween the 5′ wing segment and the 3′ wing segment, wherein eachnucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar,wherein at least one internucleoside linkage is a phosphorothioatelinkage and wherein each cytosine residue is a 5-methylcytosine.

Certain embodiments provide a modified oligonucleotide targeting apo(a)and a conjugate group, wherein the modified oligonucleotide consists of20 linked nucleosides with the nucleobase sequence of SEQ ID NO: 58,wherein the modified oligonucleotide comprises: (a) a gap segmentconsisting of ten linked deoxynucleosides; (b) a 5′ wing segmentconsisting of five linked nucleosides; (c) a 3′ wing segment consistingof five linked nucleosides; and wherein the gap segment is positionedbetween the 5′ wing segment and the 3′ wing segment, wherein eachnucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar,wherein at least one internucleoside linkage is a phosphorothioatelinkage and wherein each cytosine residue is a 5-methylcytosine.

In certain embodiments, the conjugate group is linked to the modifiedoligonucleotide at the 5′ end of the modified oligonucleotide. Incertain embodiments, the conjugate group is linked to the modifiedoligonucleotide at the 3′ end of the modified oligonucleotide.

In certain embodiments, the conjugate group comprises one or moreligands. In certain embodiments, the conjugate group comprises two ormore ligands. In certain embodiments, the conjugate group comprisesthree or more ligands. In certain embodiments, the conjugate groupcomprises three ligands. In certain embodiments, each ligand is selectedfrom among: a polysaccharide, modified polysaccharide, mannose,galactose, a mannose derivative, a galactose derivative,D-mannopyranose, L-Mannopyranose, D-Arabinose, L-Galactose,D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-Galactose,L-Galactose, α-D-Mannofuranose, β-D-Mannofuranose, α-D-Mannopyranose,β-D-Mannopyranose, α-D-Glucopyranose, β-D-Glucopyranose,α-D-Glucofuranose, β-D-Glucofuranose, α-D-fructofuranose,α-D-fructopyranose, α-D-Galactopyranose, α-D-Galactopyranose,α-D-Galactofuranose, β-D-Galactofuranose, glucosamine, sialic acid,α-D-galactosamine, N-Acetylgalactosamine,2-Amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose,2-Deoxy-2-methylamino-L-glucopyranose,4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,2-Deoxy-2-sulfoamino-D-glucopyranose, N-Glycoloyl-α-neuraminic acid,5-thio-β-D-glucopyranose, methyl2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside,4-Thio-β-D-galactopyranose, ethyl3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside,2,5-Anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose,L-4-thioribose. In certain embodiments, each ligand is N-acetylgalactosamine.

In certain embodiments, each ligand is N-acetyl galactosamine.

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises at least onephosphorus linking group or neutral linking group.

In certain embodiments, the conjugate group comprises a structureselected from among:

wherein n is from 1 to 12; and

wherein m is from 1 to 12.

In certain embodiments, the conjugate group has a tether having astructure selected from among:

-   -   wherein L is either a phosphorus linking group or a neutral        linking group;    -   Z1 is C(═O)O—R2;    -   Z2 is H, C₁-C₆ alkyl or substituted C₁-C₆ alky;    -   R2 is H, C₁-C₆ alkyl or substituted C₁-C₆ alky; and

each m1 is, independently, from 0 to 20 wherein at least one m1 isgreater than 0 for each tether.

In certain embodiments, conjugate group has a tether having a structureselected from among:

-   -   wherein Z2 is H or CH₃; and

each m1 is, independently, from 0 to 20 wherein at least one m1 isgreater than 0 for each tether.

In certain embodiments, the conjugate group has tether having astructure selected from among:

wherein n is from 1 to 12; and

wherein m is from 1 to 12.

In certain embodiments, the conjugate group is covalently attached tothe modified oligonucleotide.

In certain embodiments, the compound has a structure represented by theformula:A-B-C-D

E-F)_(q)

wherein

A is the modified oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

wherein:

A is the modified oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand;

each n is independently 0 or 1; and

q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:A-B-C

E-F)_(q)

wherein

A is the modified oligonucleotide;

B is the cleavable moiety;

C is the conjugate linker;

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:A-C-D

E-F)_(q)

wherein

A is the modified oligonucleotide;

C is the conjugate linker;

D is the branching group;

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:A-C

E-F)_(q)

wherein

A is the modified oligonucleotide;

C is the conjugate linker;

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:A-B-D

E-F)_(q)

wherein

A is the modified oligonucleotide;

B is the cleavable moiety;

D is the branching group;

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:A-B

E-F)_(q)

wherein

A is the modified oligonucleotide;

B is the cleavable moiety;

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:A-D

E-F)_(q)

wherein

A is the modified oligonucleotide;

D is the branching group;

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein each L is, independently, a phosphorus linking group or aneutral linking group; and

each n is, independently, from 1 to 20.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has the followingstructure:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker comprises a pyrrolidine. Incertain embodiments, the conjugate linker does not comprise apyrrolidine. In certain embodiments, the conjugate linker comprises PEG.In certain embodiments, the conjugate linker comprises an amide. Incertain embodiments, the conjugate linker comprises at least two amides.In certain embodiments, the conjugate linker does not comprise an amide.In certain embodiments, the conjugate linker comprises a polyamide. Incertain embodiments, the conjugate linker comprises an amine. In certainembodiments, the conjugate linker comprises one or more disulfide bonds.In certain embodiments, the conjugate linker comprises a protein bindingmoiety. In certain embodiments, the protein binding moiety comprises alipid.

In certain embodiments, the protein binding moiety is selected fromamong: cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol,geranyloxyhexyl group, hexadecylglycerol, borneol, menthol,1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl,or phenoxazine), a vitamin (e.g., folate, vitamin A, vitamin E, biotin,pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide,disaccharide, trisaccharide, tetrasaccharide, oligosaccharide,polysaccharide), an endosomolytic component, a steroid (e.g., uvaol,hecigenin, diosgenin), a terpene (e.g., triterpene, e.g.,sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid),or a cationic lipid.

In certain embodiments, the protein binding moiety is selected fromamong: a C16 to C22 long chain saturated or unsaturated fatty acid,cholesterol, cholic acid, vitamin E, adamantane or 1-pentafluoropropyl.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein each n is, independently, is from 1 to 20; and p is from 1 to 6.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein each n is, independently, from 1 to 20.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein n is from 1 to 20.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, the conjugate linker has the followingstructure:

In certain embodiments, the branching group has one of the followingstructures:

-   -   wherein each A1 is independently, O, S, C═O or NH; and    -   each n is, independently, from 1 to 20.

In certain embodiments, the branching group has one of the followingstructures:

-   -   wherein each A1 is independently, O, S, C═O or NH; and    -   each n is, independently, from 1 to 20.

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group comprises an ether.

In certain embodiments, the branching group has the following structure:

-   -   each n is, independently, from 1 to 20; and    -   m is from 2 to 6.

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group comprises:

wherein each j is an integer from 1 to 3; and

wherein each n is an integer from 1 to 20.

In certain embodiments, the branching group comprises:

In certain embodiments, each tether is selected from among:

wherein L is selected from a phosphorus linking group and a neutrallinking group;

-   -   Z1 is C(═O)O—R2;    -   Z2 is H, C1-C6 alkyl or substituted C1-C6 alky;    -   R2 is H, C1-C6 alkyl or substituted C1-C6 alky; and    -   each m1 is, independently, from 0 to 20 wherein at least one m1        is greater than 0 for each tether.

In certain embodiments, each tether is selected from among:

wherein Z2 is H or CH3; and

each m2 is, independently, from 0 to 20 wherein at least one m2 isgreater than 0 for each tether.

In certain embodiments, each tether is selected from among:

wherein n is from 1 to 12; and

wherein m is from 1 to 12.

In certain embodiments, at least one tether comprises ethylene glycol.In certain embodiments, at least one tether comprises an amide. Incertain embodiments, at least one tether comprises a polyamide. Incertain embodiments, at least one tether comprises an amine. In certainembodiments, at least two tethers are different from one another. Incertain embodiments, all of the tethers are the same as one another. Incertain embodiments, each tether is selected from among:

wherein each n is, independently, from 1 to 20; and

each p is from 1 to about 6.

In certain embodiments, each tether is selected from among:

In certain embodiments, each tether has the following structure:

-   -   wherein each n is, independently, from 1 to 20.

In certain embodiments, each tether has the following structure:

In certain embodiments, the tether has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, the tether has a structure selected from among:

In certain embodiments, the ligand is galactose. In certain embodiments,the ligand is mannose-6-phosphate.

In certain embodiments, each ligand is selected from among:

wherein each R1 is selected from OH and NHCOOH.

In certain embodiments, each ligand is selected from among:

In certain embodiments, each ligand has the following structure:

In certain embodiments, each ligand has the following structure:

In certain embodiments, the conjugate group comprises a cell-targetingmoiety.

In certain embodiments, the conjugate group comprises a cell-targetingmoiety having the following structure:

wherein each n is, independently, from 1 to 20.

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety has the followingstructure:

wherein each n is, independently, from 1 to 20.

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

wherein each Y is selected from O, S, a substituted or unsubstitutedC1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

In certain embodiments, the conjugate group comprises:

wherein each Y is selected from O, S, a substituted or unsubstitutedC1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

In certain embodiments, the conjugate group comprises:

wherein each Y is selected from O, S, a substituted or unsubstitutedC1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises a cleavable moietyselected from among: a phosphodiester, an amide, or an ester.

In certain embodiments, the conjugate group comprises a phosphodiestercleavable moiety.

In certain embodiments, the conjugate group does not comprise acleavable moiety, and wherein the conjugate group comprises aphosphorothioate linkage between the conjugate group and theoligonucleotide. In certain embodiments, the conjugate group comprisesan amide cleavable moiety. In certain embodiments, the conjugate groupcomprises an ester cleavable moiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide;

Z is H or a linked solid support; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide;

Z is H or a linked solid support; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the conjugate group comprises:

wherein Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the conjugate group comprises:

wherein Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the conjugate group comprises:

wherein Q13 is H or O(CH2)2-OCH3;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, Bx is selected from among from adenine, guanine,thymine, uracil, or cytosine, or 5-methyl cytosine. In certainembodiments, Bx is adenine. In certain embodiments, Bx is thymine. Incertain embodiments, Q13 is O(CH2)2-OCH3. In certain embodiments, Q13 isH.

In certain embodiments, the compound is in a salt form. In furtherembodiments, the compound further comprises of a pharmaceuticallyacceptable carrier or diluent. In certain embodiments, the compoundcomprises a modified oligonucleotide targeting apo(a) and a conjugategroup, or a salt thereof, and a pharmaceutically acceptable carrier ordiluent.

Certain embodiments provide a composition comprising a conjugatedantisense compound as described herein, wherein the viscosity level ofthe compound is less than 40 centipoise (cP). In certain embodiments,the conjugated antisense compounds as described herein are efficaciousby virtue of having a viscosity of less than 40 cP, less than 35 cP,less than 30 cP, less than 25 cP, less than 20 cP or less than 15 cPwhen measured by the parameters as described in Example 125.

Certain embodiments provide compositions and methods comprisingadministering to an animal a conjugated antisense compound orcomposition disclosed herein. In certain embodiments, administering theconjugated antisense compound prevents, treats, ameliorates, or slowsprogression of a cardiovascular, metabolic and/or inflammatory disease.

Certain embodiments provide compositions and methods for use in therapyto treat an apo(a) related disease, disorder or condition. Certainembodiments provide compositions and methods for use in therapy to treatan Lp(a) related disease, disorder or condition. In certain embodiments,apo(a) and/or Lp(a) levels are elevated in an animal. In certainembodiments, the composition is a compound comprising an apo(a) specificinhibitor. In certain embodiments, the apo(a) specific inhibitor is anucleic acid. In certain embodiments, the nucleic acid is an antisensecompound. In certain embodiments, the antisense compound is a modifiedoligonucleotide targeting apo(a). In certain embodiments, the antisensecompound is a modified oligonucleotide targeting apo(a) and a conjugategroup. In certain embodiments, the modified oligonucleotide targetingapo(a) with the conjugate group, is used in treating, preventing,slowing progression, ameliorating a cardiovascular and/or metabolicdisease, disorder or condition. In certain embodiments, the compositionsand methods for therapy include administering an apo(a) specificinhibitor to an individual in need thereof.

Certain embodiments provide compositions and methods for reducing apo(a)levels. Certain embodiments provide compositions and methods forreducing Lp(a) levels. In certain embodiments, reducing apo(a) levels ina tissue, organ or subject improves the ratio of LDL to HDL or the ratioof TG to HDL. Certain embodiments provide compositions and methods toreduce apo(a) mRNA or protein expression in an animal comprisingadministering to the animal a conjugated antisense compound orcomposition disclosed herein to reduce apo(a) mRNA or protein expressionin the animal. Certain embodiments provide compositions and methods toreduce Lp(a) levels in an animal comprising administering to the animala conjugated antisense compound or composition disclosed herein toreduce apo(a) mRNA or protein expression in the animal.

Certain embodiments provide compositions and methods for preventing,treating, delaying, slowing the progression and/or ameliorating apo(a)related diseases, disorders, and conditions in a subject in needthereof. Certain embodiments provide compositions and methods forpreventing, treating, delaying, slowing the progression and/orameliorating Lp(a) related diseases, disorders, and conditions in asubject in need thereof. In certain embodiments, such diseases,disorders, and conditions include inflammatory, cardiovascular and/ormetabolic diseases, disorders, and conditions. Certain suchcardiovascular diseases, disorders or conditions include, but are notlimited to, aortic stenosis, aneurysm (e.g., abdominal aortic aneurysm),angina, arrhythmia, atherosclerosis, cerebrovascular disease, coronaryartery disease, coronary heart disease, dyslipidemia,hypercholesterolemia, hyperlipidemia, hypertension,hypertriglyceridemia, myocardial infarction, peripheral vascular disease(e.g., peripheral artery disease, peripheral artery occlusive disease),retinal vascular occlusion, or stroke. Certain such metabolic diseases,disorders or conditions include, but are not limited to, hyperglycemia,prediabetes, diabetes (type I and type II), obesity, insulin resistance,metabolic syndrome and diabetic dyslipidemia. Certain such inflammatorydiseases, disorders or conditions include, but are not limited to,aortic stenosis, coronary artery disease (CAD), Alzheimer's Disease andthromboembolic diseases, disorder or conditions. Certain thromboembolicdiseases, disorders or conditions include, but are not limited to,stroke, thrombosis (e.g., venous thromboembolism), myocardial infarctionand peripheral vascular disease. Certain embodiments providecompositions and methods for preventing, treating, delaying, slowing theprogression and/or ameliorating aortic stenosis.

Certain embodiments provide a method of reducing at least one symptom ofa cardiovascular disease, disorder or condition. In certain embodiments,the symptoms include, but are not limited to, angina, chest pain,shortness of breath, palpitations, weakness, dizziness, nausea,sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation,swelling in the lower extremities, cyanosis, fatigue, fainting, numbnessof the face, numbness of the limbs, claudication or cramping of muscles,bloating of the abdomen, and fever. Certain embodiments provide a methodof reducing at least one symptom of aortic stenosis.

In certain embodiments, the modulation of apo(a) or Lp(a) expressionoccurs in a cell, tissue or organ. In certain embodiments, themodulations occur in a cell, tissue or organ in an animal. In certainembodiments, the modulation is a reduction in apo(a) mRNA level. Incertain embodiments, the modulation is a reduction in apo(a) proteinlevel. In certain embodiments, both apo(a) mRNA and protein levels arereduced. In certain embodiments, the modulation is a reduction in Lp(a)level. Such reduction may occur in a time-dependent or in adose-dependent manner.

In certain embodiments, the subject or animal is human.

In certain embodiments, the conjugated antisense compound isparenterally administered. In further embodiments, the parenteraladministration is subcutaneous.

In certain embodiments, the conjugated antisense compound or compositionis co-administered with a second agent or therapy. In certainembodiments, the conjugated antisense compound or composition and thesecond agent are administered concomitantly.

In certain embodiments, the second agent is a glucose-lowering agent. Incertain embodiments, the second agent is a LDL, TG or cholesterollowering agent. In certain embodiments, the second agent is ananti-inflammatory agent. In certain embodiments, the second agent is anAlzheimer Disease drug. In certain embodiments, the second agent can be,but is not limited to, a non-steroidal anti-inflammatory drug (NSAIDe.g., aspirin), niacin (e.g., Niaspan), nicotinic acid, an apoBinhibitor (e.g., Mipomersen), a CETP inhibitor (e.g., Anacetrapib), anapo(a) inhibitor, a thyroid hormone analog (e.g., Eprotirome), a HMG-CoAreductase inhibitor (e.g., a statin), a fibrate (e.g., Gemfibrozil) andan microsomal triglyceride transfer protein inhibitor (e.g.,Lomitapide). The therapy can be, but is not limited to, Lp(a) apheresis.Agents or therapies can be co-administered or administeredconcomitantly. Agents or therapies can be sequentially or subsequentlyadministered.

Certain embodiments provide use of a conjugated antisense compoundtargeted to apo(a) for decreasing apo(a) levels in an animal. Certainembodiments provide use of a conjugated antisense compound targeted toapo(a) for decreasing Lp(a) levels in an animal. Certain embodimentsprovide use of a conjugated antisense compounds targeted to apo(a) forthe treatment, prevention, or amelioration of a disease, disorder, orcondition associated with apo(a). Certain embodiments provide use of aconjugated antisense compounds targeted to apo(a) for the treatment,prevention, or amelioration of a disease, disorder, or conditionassociated with Lp(a).

Certain embodiments provide use of a conjugated antisense compoundtargeted to apo(a) in the preparation of a medicament for decreasingapo(a) levels in an animal. Certain embodiments provide use of aconjugated antisense compound targeted to apo(a) in the preparation of amedicament for decreasing Lp(a) levels in an animal. Certain embodimentsprovide use of a conjugated antisense compound for the preparation of amedicament for the treatment, prevention, or amelioration of a disease,disorder, or condition associated with apo(a). Certain embodimentsprovide use of a conjugated antisense compound for the preparation of amedicament for the treatment, prevention, or amelioration of a disease,disorder, or condition associated with Lp(a).

Certain embodiments provide the use of a conjugated antisense compoundas described herein in the manufacture of a medicament for treating,ameliorating, delaying or preventing one or more of a disease related toapo(a) and/or Lp(a).

Certain embodiments provide a kit for treating, preventing, orameliorating a disease, disorder or condition as described hereinwherein the kit comprises: (i) an apo(a) specific inhibitor as describedherein; and optionally (ii) a second agent or therapy as describedherein.

A kit of the present invention can further include instructions forusing the kit to treat, prevent, or ameliorate a disease, disorder orcondition as described herein by combination therapy as describedherein.

B. Certain Compounds

In certain embodiments, the invention provides conjugated antisensecompounds comprising antisense oligonucleotides and a conjugate.

a. Certain Antisense Oligonucleotides

In certain embodiments, the invention provides antisenseoligonucleotides. Such antisense oligonucleotides comprise linkednucleosides, each nucleoside comprising a sugar moiety and a nucleobase.The structure of such antisense oligonucleotides may be considered interms of chemical features (e.g., modifications and patterns ofmodifications) and nucleobase sequence (e.g., sequence of antisenseoligonucleotide, identify and sequence of target nucleic acid).

i. Certain Chemistry Features

In certain embodiments, antisense oligonucleotide comprise one or moremodification. In certain such embodiments, antisense oligonucleotidescomprise one or more modified nucleosides and/or modifiedinternucleoside linkages. In certain embodiments, modified nucleosidescomprise a modified sugar moiety and/or modified nucleobase.

1. Certain Sugar Moieties

In certain embodiments, compounds of the disclosure comprise one or moremodified nucleosides comprising a modified sugar moiety. Such compoundscomprising one or more sugar-modified nucleosides may have desirableproperties, such as enhanced nuclease stability or increased bindingaffinity with a target nucleic acid relative to an oligonucleotidecomprising only nucleosides comprising naturally occurring sugarmoieties. In certain embodiments, modified sugar moieties aresubstituted sugar moieties. In certain embodiments, modified sugarmoieties are sugar surrogates. Such sugar surrogates may comprise one ormore substitutions corresponding to those of substituted sugar moieties.

In certain embodiments, modified sugar moieties are substituted sugarmoieties comprising one or more non-bridging sugar substituent,including but not limited to substituents at the 2′ and/or 5′ positions.Examples of sugar substituents suitable for the 2′-position, include,but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and2′-O(CH₂)₂OCH₃ (“MOE”). In certain embodiments, sugar substituents atthe 2′ position is selected from allyl, amino, azido, thio, O-allyl,O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl; OCF₃, O(CH₂)₂SCH₃,O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where each Rm and Rn is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. Examplesof sugar substituents at the 5′-position, include, but are not limitedto: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In certainembodiments, substituted sugars comprise more than one non-bridgingsugar substituent, for example, 2′-F-5′-methyl sugar moieties (see,e.g., PCT International Application WO 2008/101157, for additional5′,2′-bis substituted sugar moieties and nucleosides).

Nucleosides comprising 2′-substituted sugar moieties are referred to as2′-substituted nucleosides. In certain embodiments, a 2′-substitutednucleoside comprises a 2′-substituent group selected from halo, allyl,amino, azido, SH, CN, OCN, CF₃, OCF₃, O, S, or N(R_(m))-alkyl; O, S, orN(R_(m))-alkenyl; O, S or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl,alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H, an amino protecting group orsubstituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituent groupscan be further substituted with one or more substituent groupsindependently selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,alkenyl and alkynyl.

In certain embodiments, a 2′-substituted nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃,OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂,and O—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, O—CH₃, andOCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. Incertain such embodiments, the bicyclic sugar moiety comprises a bridgebetween the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′sugar substituents, include, but are not limited to:—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—; 4′-CH₂-2′,4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′; 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′;4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and 4′-CH(CH₂OCH₃)—O-2′, andanalogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15,2008); 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g.,WO2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ and analogsthereof (see, e.g., WO2008/150729, published Dec. 11, 2008);4′-CH₂—O—N(CH₃)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004);4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′-, wherein each R is,independently, H, a protecting group, or C₁-C₁₂ alkyl; 4′-CH₂—N(R)—O-2′,wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see, U.S. Pat. No.7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g.,Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′ and analogs thereof (see, published PCT InternationalApplication WO 2008/154401, published on Dec. 8, 2008).

In certain embodiments, such 4′ to 2′ bridges independently comprisefrom 1 to 4 linked groups independently selected from—[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—,—C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and—N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA,(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt),(G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino(4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA,and (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, aprotecting group, or C₁-C₁₂ alkyl.

Additional bicyclic sugar moieties are known in the art, for example:Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63,10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379(Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr.Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207,6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO2007/134181; U.S. Patent Publication Nos. US2004/0171570,US2007/0287831, and US2008/0039618; U.S. patent Ser. Nos. 12/129,154,60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787,and 61/099,844; and PCT International Applications Nos.PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.

In certain embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) bicyclicnucleosides have been incorporated into antisense oligonucleotides thatshowed antisense activity (Frieden et al., Nucleic Acids Research, 2003,21, 6365-6372).

In certain embodiments, substituted sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCTInternational Application WO 2007/134181, published on Nov. 22, 2007,wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinylgroup).

In certain embodiments, modified sugar moieties are sugar surrogates. Incertain such embodiments, the oxygen atom of the naturally occurringsugar is substituted, e.g., with a sulfur, carbon or nitrogen atom. Incertain such embodiments, such modified sugar moiety also comprisesbridging and/or non-bridging substituents as described above. Forexample, certain sugar surrogates comprise a 4′-sulfur atom and asubstitution at the 2′-position (see, e.g., published U.S. PatentApplication US2005/0130923, published on Jun. 16, 2005) and/or the 5′position. By way of additional example, carbocyclic bicyclic nucleosideshaving a 4′-2′ bridge have been described (see, e.g., Freier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740).

In certain embodiments, sugar surrogates comprise rings having otherthan 5-atoms. For example, in certain embodiments, a sugar surrogatecomprises a morphlino. Morpholino compounds and their use in oligomericcompounds has been reported in numerous patents and published articles(see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; andU.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As usedhere, the term “morpholino” means a sugar surrogate having the followingstructure:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

For another example, in certain embodiments, a sugar surrogate comprisesa six-membered tetrahydropyran. Such tetrahydropyrans may be furthermodified or substituted. Nucleosides comprising such modifiedtetrahydropyrans include, but are not limited to, hexitol nucleic acid(HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA(F-HNA), and those compounds having Formula VI:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula VI:

Bx is a nucleobase moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the antisense compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, or substituted C₂-C₆ alkynyl; and

each of R₁ and R₂ is independently selected from among: hydrogen,halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and eachJ₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VI areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH.

In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ ismethyl. In certain embodiments, THP nucleosides of Formula VI areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds (see, e.g., review article:Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 Published on Aug. 21, 2008 for otherdisclosed 5′,2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

In certain embodiments, the present disclosure provides oligonucleotidescomprising modified nucleosides. Those modified nucleotides may includemodified sugars, modified nucleobases, and/or modified linkages. Thespecific modifications are selected such that the resultingoligonucleotides possess desirable characteristics. In certainembodiments, oligonucleotides comprise one or more RNA-like nucleosides.In certain embodiments, oligonucleotides comprise one or more DNA-likenucleotides.

2. Certain Nucleobase Modifications

In certain embodiments, nucleosides of the present disclosure compriseone or more unmodified nucleobases. In certain embodiments, nucleosidesof the present disclosure comprise one or more modified nucleobases.

In certain embodiments, modified nucleobases are selected from:universal bases, hydrophobic bases, promiscuous bases, size-expandedbases, and fluorinated bases as defined herein. 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine;5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Further modified nucleobasesinclude tricyclic pyrimidines such as phenoxazinecytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz,J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613; and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, Crooke, S. T. and Lebleu, B., Eds., CRCPress, 1993, 273-288.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include without limitation, U.S. Pat. Nos.3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985;5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference in its entirety.

3. Certain Internucleoside Linkages

In certain embodiments, the present disclosure provides oligonucleotidescomprising linked nucleosides. In such embodiments, nucleosides may belinked together using any internucleoside linkage. The two main classesof internucleoside linking groups are defined by the presence or absenceof a phosphorus atom. Representative phosphorus containinginternucleoside linkages include, but are not limited to,phosphodiesters (PO), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (PS). Representativenon-phosphorus containing internucleoside linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—),thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane(—O—Si(H)₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—).Modified linkages, compared to natural phosphodiester linkages, can beused to alter, typically increase, nuclease resistance of theoligonucleotide. In certain embodiments, internucleoside linkages havinga chiral atom can be prepared as a racemic mixture, or as separateenantiomers. Representative chiral linkages include, but are not limitedto, alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing internucleosidelinkages are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), a or b such as for sugar anomers, or as(D) or (L) such as for amino acids etc. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

Neutral internucleoside linkages include without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

4. Certain Motifs

In certain embodiments, antisense oligonucleotides comprise one or moremodified nucleoside (e.g., nucleoside comprising a modified sugar and/ormodified nucleobase) and/or one or more modified internucleosidelinkage. The pattern of such modifications on an oligonucleotide isreferred to herein as a motif. In certain embodiments, sugar,nucleobase, and linkage motifs are independent of one another.

a. Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type ofmodified sugar moieties and/or naturally occurring sugar moietiesarranged along an oligonucleotide or region thereof in a defined patternor sugar modification motif. Such motifs may include any of the sugarmodifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of aregion having a gapmer sugar motif, which comprises two external regionsor “wings” and a central or internal region or “gap.” The three regionsof a gapmer sugar motif (the 5′-wing, the gap, and the 3′-wing) form acontiguous sequence of nucleosides wherein at least some of the sugarmoieties of the nucleosides of each of the wings differ from at leastsome of the sugar moieties of the nucleosides of the gap. Specifically,at least the sugar moieties of the nucleosides of each wing that areclosest to the gap (the 3′-most nucleoside of the 5′-wing and the5′-most nucleoside of the 3′-wing) differ from the sugar moiety of theneighboring gap nucleosides, thus defining the boundary between thewings and the gap. In certain embodiments, the sugar moieties within thegap are the same as one another. In certain embodiments, the gapincludes one or more nucleoside having a sugar moiety that differs fromthe sugar moiety of one or more other nucleosides of the gap. In certainembodiments, the sugar motifs of the two wings are the same as oneanother (symmetric sugar gapmer). In certain embodiments, the sugarmotifs of the 5′-wing differs from the sugar motif of the 3′-wing(asymmetric sugar gapmer).

i. Certain 5′-Wings

In certain embodiments, the 5′-wing of a gapmer consists of 1 to 8linked nucleosides. In certain embodiments, the 5′-wing of a gapmerconsists of 1 to 7 linked nucleosides. In certain embodiments, the5′-wing of a gapmer consists of 1 to 6 linked nucleosides. In certainembodiments, the 5′-wing of a gapmer consists of 1 to 5 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of2 to 5 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 3 to 5 linked nucleosides. In certain embodiments,the 5′-wing of a gapmer consists of 4 or 5 linked nucleosides. Incertain embodiments, the 5′-wing of a gapmer consists of 1 to 4 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of1 to 3 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 1 or 2 linked nucleosides. In certain embodiments,the 5′-wing of a gapmer consists of 2 to 4 linked nucleosides. Incertain embodiments, the 5′-wing of a gapmer consists of 2 or 3 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of3 or 4 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 1 nucleoside. In certain embodiments, the 5′-wing ofa gapmer consists of 2 linked nucleosides. In certain embodiments, the5′-wing of a gapmer consists of 3 linked nucleosides. In certainembodiments, the 5′-wing of a gapmer consists of 4 linked nucleosides.In certain embodiments, the 5′-wing of a gapmer consists of 5 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of6 linked nucleosides.

In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside. In certain embodiments, the 5′-wing of a gapmercomprises at least two bicyclic nucleosides. In certain embodiments, the5′-wing of a gapmer comprises at least three bicyclic nucleosides. Incertain embodiments, the 5′-wing of a gapmer comprises at least fourbicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmercomprises at least one constrained ethyl nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one LNAnucleoside. In certain embodiments, each nucleoside of the 5′-wing of agapmer is a bicyclic nucleoside. In certain embodiments, each nucleosideof the 5′-wing of a gapmer is a constrained ethyl nucleoside. In certainembodiments, each nucleoside of the 5′-wing of a gapmer is a LNAnucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least onenon-bicyclic modified nucleoside. In certain embodiments, the 5′-wing ofa gapmer comprises at least one 2′-substituted nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one 2′-MOEnucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one 2′-OMe nucleoside. In certain embodiments, each nucleoside ofthe 5′-wing of a gapmer is a non-bicyclic modified nucleoside. Incertain embodiments, each nucleoside of the 5′-wing of a gapmer is a2′-substituted nucleoside. In certain embodiments, each nucleoside ofthe 5′-wing of a gapmer is a 2′-MOE nucleoside. In certain embodiments,each nucleoside of the 5′-wing of a gapmer is a 2′-OMe nucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least one2′-deoxynucleoside. In certain embodiments, each nucleoside of the5′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments,the 5′-wing of a gapmer comprises at least one ribonucleoside. Incertain embodiments, each nucleoside of the 5′-wing of a gapmer is aribonucleoside. In certain embodiments, one, more than one, or each ofthe nucleosides of the 5′-wing is an RNA-like nucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one non-bicyclic modified nucleoside.In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-substituted nucleoside. Incertain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-MOE nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one bicyclicnucleoside and at least one 2′-OMe nucleoside. In certain embodiments,the 5′-wing of a gapmer comprises at least one bicyclic nucleoside andat least one 2′-deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least oneconstrained ethyl nucleoside and at least one non-bicyclic modifiednucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-substitutednucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-MOEnucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-OMenucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one2′-deoxynucleoside.

ii. Certain 3′-Wings

In certain embodiments, the 3′-wing of a gapmer consists of 1 to 8linked nucleosides. In certain embodiments, the 3′-wing of a gapmerconsists of 1 to 7 linked nucleosides. In certain embodiments, the3′-wing of a gapmer consists of 1 to 6 linked nucleosides. In certainembodiments, the 3′-wing of a gapmer consists of 1 to 5 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of2 to 5 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 3 to 5 linked nucleosides. In certain embodiments,the 3′-wing of a gapmer consists of 4 or 5 linked nucleosides. Incertain embodiments, the 3′-wing of a gapmer consists of 1 to 4 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of1 to 3 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 1 or 2 linked nucleosides. In certain embodiments,the 3′-wing of a gapmer consists of 2 to 4 linked nucleosides. Incertain embodiments, the 3′-wing of a gapmer consists of 2 or 3 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of3 or 4 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 1 nucleoside. In certain embodiments, the 3′-wing ofa gapmer consists of 2 linked nucleosides. In certain embodiments, the3′-wing of a gapmer consists of 3 linked nucleosides. In certainembodiments, the 3′-wing of a gapmer consists of 4 linked nucleosides.In certain embodiments, the 3′-wing of a gapmer consists of 5 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of6 linked nucleosides.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside. In certain embodiments, each nucleoside of the 3′-wing of agapmer is a bicyclic nucleoside. In certain embodiments, each nucleosideof the 3′-wing of a gapmer is a constrained ethyl nucleoside. In certainembodiments, each nucleoside of the 3′-wing of a gapmer is a LNAnucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onenon-bicyclic modified nucleoside. In certain embodiments, the 3′-wing ofa gapmer comprises at least two non-bicyclic modified nucleosides. Incertain embodiments, the 3′-wing of a gapmer comprises at least threenon-bicyclic modified nucleosides. In certain embodiments, the 3′-wingof a gapmer comprises at least four non-bicyclic modified nucleosides.In certain embodiments, the 3′-wing of a gapmer comprises at least one2′-substituted nucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one 2′-MOE nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one 2′-OMe nucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is anon-bicyclic modified nucleoside. In certain embodiments, eachnucleoside of the 3′-wing of a gapmer is a 2′-substituted nucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is a2′-MOE nucleoside. In certain embodiments, each nucleoside of the3′-wing of a gapmer is a 2′-OMe nucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least one2′-deoxynucleoside. In certain embodiments, each nucleoside of the3′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments,the 3′-wing of a gapmer comprises at least one ribonucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is aribonucleoside. In certain embodiments, one, more than one, or each ofthe nucleosides of the 5′-wing is an RNA-like nucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one non-bicyclic modified nucleoside.In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-substituted nucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-MOE nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one bicyclicnucleoside and at least one 2′-OMe nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one bicyclic nucleoside andat least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least oneconstrained ethyl nucleoside and at least one non-bicyclic modifiednucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-substitutednucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-MOEnucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-OMenucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least oneLNA nucleoside and at least one non-bicyclic modified nucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside and at least one 2′-substituted nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside and at least one 2′-MOE nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside and atleast one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one LNA nucleoside and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one non-bicyclic modified nucleoside, andat least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing ofa gapmer comprises at least one constrained ethyl nucleoside, at leastone non-bicyclic modified nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one LNA nucleoside, at least one non-bicyclicmodified nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-substituted nucleoside, and atleast one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one constrained ethyl nucleoside, at least one2′-substituted nucleoside, and at least one 2′-deoxynucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside, at least one 2′-substituted nucleoside, and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-MOE nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside, at least one 2′-MOEnucleoside, and at least one 2′-deoxynucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside, at leastone 2′-MOE nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-OMe nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside, at least one 2′-OMenucleoside, and at least one 2′-deoxynucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside, at leastone 2′-OMe nucleoside, and at least one 2′-deoxynucleoside.

iii. Certain Central Regions (Gaps)

In certain embodiments, the gap of a gapmer consists of 6 to 20 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 6to 15 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 6 to 12 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 6 to 10 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 6 to 9 linked nucleosides.In certain embodiments, the gap of a gapmer consists of 6 to 8 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 6or 7 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 7 to 10 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 7 to 9 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 7 or 8 linked nucleosides.In certain embodiments, the gap of a gapmer consists of 8 to 10 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 8or 9 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 6 linked nucleosides. In certain embodiments, the gap of agapmer consists of 7 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 8 linked nucleosides. In certain embodiments,the gap of a gapmer consists of 9 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 10 linked nucleosides. Incertain embodiments, the gap of a gapmer consists of 11 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 12linked nucleosides.

In certain embodiments, each nucleoside of the gap of a gapmer is a2′-deoxynucleoside. In certain embodiments, the gap comprises one ormore modified nucleosides. In certain embodiments, each nucleoside ofthe gap of a gapmer is a 2′-deoxynucleoside or is a modified nucleosidethat is “DNA-like.” In such embodiments, “DNA-like” means that thenucleoside has similar characteristics to DNA, such that a duplexcomprising the gapmer and an RNA molecule is capable of activating RNaseH. For example, under certain conditions, 2′-(ara)-F have been shown tosupport RNase H activation, and thus is DNA-like. In certainembodiments, one or more nucleosides of the gap of a gapmer is not a2′-deoxynucleoside and is not DNA-like. In certain such embodiments, thegapmer nonetheless supports RNase H activation (e.g., by virtue of thenumber or placement of the non-DNA nucleosides).

In certain embodiments, gaps comprise a stretch of unmodified2′-deoxynucleoside interrupted by one or more modified nucleosides, thusresulting in three sub-regions (two stretches of one or more2′-deoxynucleosides and a stretch of one or more interrupting modifiednucleosides). In certain embodiments, no stretch of unmodified2′-deoxynucleosides is longer than 5, 6, or 7 nucleosides. In certainembodiments, such short stretches is achieved by using short gapregions. In certain embodiments, short stretches are achieved byinterrupting a longer gap region.

In certain embodiments, the gap comprises one or more modifiednucleosides. In certain embodiments, the gap comprises one or moremodified nucleosides selected from among cEt, FHNA, LNA, and2-thio-thymidine. In certain embodiments, the gap comprises one modifiednucleoside. In certain embodiments, the gap comprises a 5′-substitutedsugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certainembodiments, the gap comprises two modified nucleosides. In certainembodiments, the gap comprises three modified nucleosides. In certainembodiments, the gap comprises four modified nucleosides. In certainembodiments, the gap comprises two or more modified nucleosides and eachmodified nucleoside is the same. In certain embodiments, the gapcomprises two or more modified nucleosides and each modified nucleosideis different.

In certain embodiments, the gap comprises one or more modified linkages.In certain embodiments, the gap comprises one or more methyl phosphonatelinkages. In certain embodiments the gap comprises two or more modifiedlinkages. In certain embodiments, the gap comprises one or more modifiedlinkages and one or more modified nucleosides. In certain embodiments,the gap comprises one modified linkage and one modified nucleoside. Incertain embodiments, the gap comprises two modified linkages and two ormore modified nucleosides.

b. Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modifiedinternucleoside linkages arranged along the oligonucleotide or regionthereof in a defined pattern or modified internucleoside linkage motif.In certain embodiments, oligonucleotides comprise a region having analternating internucleoside linkage motif. In certain embodiments,oligonucleotides of the present disclosure comprise a region ofuniformly modified internucleoside linkages. In certain suchembodiments, the oligonucleotide comprises a region that is uniformlylinked by phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide is uniformly linked by phosphorothioateinternucleoside linkages. In certain embodiments, each internucleosidelinkage of the oligonucleotide is selected from phosphodiester andphosphorothioate. In certain embodiments, each internucleoside linkageof the oligonucleotide is selected from phosphodiester andphosphorothioate and at least one internucleoside linkage isphosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 7 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least8 phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 9 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least 11 phosphorothioateinternucleoside linkages. In certain embodiments, the oligonucleotidecomprises at least 12 phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least 13phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 14 phosphorothioate internucleosidelinkages.

In certain embodiments, the oligonucleotide comprises at least one blockof at least 6 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 7 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 9 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least block of atleast one 12 consecutive phosphorothioate internucleoside linkages. Incertain such embodiments, at least one such block is located at the 3′end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide. In certain embodiments, the oligonucleotide comprisesless than 15 phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises less than 14 phosphorothioateinternucleoside linkages. In certain embodiments, the oligonucleotidecomprises less than 13 phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises less than 12phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises less than 11 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises lessthan 10 phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises less than 9 phosphorothioateinternucleoside linkages. In certain embodiments, the oligonucleotidecomprises less than 8 phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises less than 7phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises less than 6 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises lessthan 5 phosphorothioate internucleoside linkages.

c. Certain Nucleobase Modification Motifs

In certain embodiments, oligonucleotides comprise chemical modificationsto nucleobases arranged along the oligonucleotide or region thereof in adefined pattern or nucleobases modification motif. In certain suchembodiments, nucleobase modifications are arranged in a gapped motif. Incertain embodiments, nucleobase modifications are arranged in analternating motif. In certain embodiments, each nucleobase is modified.In certain embodiments, none of the nucleobases is chemically modified.

In certain embodiments, oligonucleotides comprise a block of modifiednucleobases. In certain such embodiments, the block is at the 3′-end ofthe oligonucleotide. In certain embodiments the block is within 3nucleotides of the 3′-end of the oligonucleotide. In certain suchembodiments, the block is at the 5′-end of the oligonucleotide. Incertain embodiments the block is within 3 nucleotides of the 5′-end ofthe oligonucleotide.

In certain embodiments, nucleobase modifications are a function of thenatural base at a particular position of an oligonucleotide. Forexample, in certain embodiments each purine or each pyrimidine in anoligonucleotide is modified. In certain embodiments, each adenine ismodified. In certain embodiments, each guanine is modified. In certainembodiments, each thymine is modified. In certain embodiments, eachcytosine is modified. In certain embodiments, each uracil is modified.

In certain embodiments, some, all, or none of the cytosine moieties inan oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methylcytosine is not a “modified nucleobase.” Accordingly, unless otherwiseindicated, unmodified nucleobases include both cytosine residues havinga 5-methyl and those lacking a 5 methyl. In certain embodiments, themethylation state of all or some cytosine nucleobases is specified.

In certain embodiments, chemical modifications to nucleobases compriseattachment of certain conjugate groups to nucleobases. In certainembodiments, each purine or each pyrimidine in an oligonucleotide may beoptionally modified to comprise a conjugate group.

d. Certain Overall Lengths

In certain embodiments, the present disclosure provides oligonucleotidesof any of a variety of ranges of lengths. In certain embodiments,oligonucleotides consist of X to Y linked nucleosides, where Xrepresents the fewest number of nucleosides in the range and Yrepresents the largest number of nucleosides in the range. In certainsuch embodiments, X and Y are each independently selected from 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, and 50; provided that X≤Y. For example, in certainembodiments, the oligonucleotide may consist of 8 to 9, 8 to 10, 8 to11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to26, 11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to30 linked nucleosides. In embodiments where the number of nucleosides ofan oligonucleotide of a compound is limited, whether to a range or to aspecific number, the compound may, nonetheless further compriseadditional other substituents. For example, an oligonucleotidecomprising 8-30 nucleosides excludes oligonucleotides having 31nucleosides, but, unless otherwise indicated, such an oligonucleotidemay further comprise, for example one or more conjugate groups, terminalgroups, or other substituents.

Further, where an oligonucleotide is described by an overall lengthrange and by regions having specified lengths, and where the sum ofspecified lengths of the regions is less than the upper limit of theoverall length range, the oligonucleotide may have additionalnucleosides, beyond those of the specified regions, provided that thetotal number of nucleosides does not exceed the upper limit of theoverall length range.

5. Certain Antisense Oligonucleotide Chemistry Motifs

In certain embodiments, the chemical structural features of antisenseoligonucleotides are characterized by their sugar motif, internucleosidelinkage motif, nucleobase modification motif and overall length. Incertain embodiments, such parameters are each independent of oneanother. Thus, each internucleoside linkage of an oligonucleotide havinga gapmer sugar motif may be modified or unmodified and may or may notfollow the gapmer modification pattern of the sugar modifications. Thus,the internucleoside linkages within the wing regions of a sugar-gapmermay be the same or different from one another and may be the same ordifferent from the internucleoside linkages of the gap region. Likewise,such sugar-gapmer oligonucleotides may comprise one or more modifiednucleobase independent of the gapmer pattern of the sugar modifications.One of skill in the art will appreciate that such motifs may be combinedto create a variety of oligonucleotides.

In certain embodiments, the selection of internucleoside linkage andnucleoside modification are not independent of one another.

i. Certain Sequences and Targets

In certain embodiments, the invention provides antisenseoligonucleotides having a sequence complementary to a target nucleicacid. Such antisense compounds are capable of hybridizing to a targetnucleic acid, resulting in at least one antisense activity. In certainembodiments, antisense compounds specifically hybridize to one or moretarget nucleic acid. In certain embodiments, a specifically hybridizingantisense compound has a nucleobase sequence comprising a region havingsufficient complementarity to a target nucleic acid to allowhybridization and result in antisense activity and insufficientcomplementarity to any non-target so as to avoid or reduce non-specifichybridization to non-target nucleic acid sequences under conditions inwhich specific hybridization is desired (e.g., under physiologicalconditions for in vivo or therapeutic uses, and under conditions inwhich assays are performed in the case of in vitro assays). In certainembodiments, oligonucleotides are selective between a target andnon-target, even though both target and non-target comprise the targetsequence. In such embodiments, selectivity may result from relativeaccessibility of the target region of one nucleic acid molecule comparedto the other.

In certain embodiments, the present disclosure provides antisensecompounds comprising oligonucleotides that are fully complementary tothe target nucleic acid over the entire length of the oligonucleotide.In certain embodiments, oligonucleotides are 99% complementary to thetarget nucleic acid. In certain embodiments, oligonucleotides are 95%complementary to the target nucleic acid. In certain embodiments, sucholigonucleotides are 90% complementary to the target nucleic acid.

In certain embodiments, such oligonucleotides are 85% complementary tothe target nucleic acid. In certain embodiments, such oligonucleotidesare 80% complementary to the target nucleic acid. In certainembodiments, an antisense compound comprises a region that is fullycomplementary to a target nucleic acid and is at least 80% complementaryto the target nucleic acid over the entire length of theoligonucleotide. In certain such embodiments, the region of fullcomplementarity is from 6 to 14 nucleobases in length.

In certain embodiments, oligonucleotides comprise a hybridizing regionand a terminal region. In certain such embodiments, the hybridizingregion consists of 12-30 linked nucleosides and is fully complementaryto the target nucleic acid. In certain embodiments, the hybridizingregion includes one mismatch relative to the target nucleic acid. Incertain embodiments, the hybridizing region includes two mismatchesrelative to the target nucleic acid. In certain embodiments, thehybridizing region includes three mismatches relative to the targetnucleic acid. In certain embodiments, the terminal region consists of1-4 terminal nucleosides. In certain embodiments, the terminalnucleosides are at the 3′ end. In certain embodiments, one or more ofthe terminal nucleosides are not complementary to the target nucleicacid.

Antisense mechanisms include any mechanism involving the hybridizationof an oligonucleotide with target nucleic acid, wherein thehybridization results in a biological effect. In certain embodiments,such hybridization results in either target nucleic acid degradation oroccupancy with concomitant inhibition or stimulation of the cellularmachinery involving, for example, translation, transcription, orsplicing of the target nucleic acid.

One type of antisense mechanism involving degradation of target RNA isRNase H mediated antisense. RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. It is known in the art thatsingle-stranded antisense compounds which are “DNA-like” elicit RNase Hactivity in mammalian cells. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof DNA-like oligonucleotide-mediated inhibition of gene expression.

In certain embodiments, a conjugate group comprises a cleavable moiety.In certain embodiments, a conjugate group comprises one or morecleavable bond. In certain embodiments, a conjugate group comprises alinker. In certain embodiments, a linker comprises a protein bindingmoiety. In certain embodiments, a conjugate group comprises acell-targeting moiety (also referred to as a cell-targeting group). Incertain embodiments a cell-targeting moiety comprises a branching group.In certain embodiments, a cell-targeting moiety comprises one or moretethers. In certain embodiments, a cell-targeting moiety comprises acarbohydrate or carbohydrate cluster.

ii. Certain Cleavable Moieties

In certain embodiments, a cleavable moiety is a cleavable bond. Incertain embodiments, a cleavable moiety comprises a cleavable bond. Incertain embodiments, the conjugate group comprises a cleavable moiety.In certain such embodiments, the cleavable moiety attaches to theantisense oligonucleotide. In certain such embodiments, the cleavablemoiety attaches directly to the cell-targeting moiety. In certain suchembodiments, the cleavable moiety attaches to the conjugate linker. Incertain embodiments, the cleavable moiety comprises a phosphate orphosphodiester. In certain embodiments, the cleavable moiety is acleavable nucleoside or nucleoside analog. In certain embodiments, thenucleoside or nucleoside analog comprises an optionally protectedheterocyclic base selected from a purine, substituted purine, pyrimidineor substituted pyrimidine. In certain embodiments, the cleavable moietyis a nucleoside comprising an optionally protected heterocyclic baseselected from uracil, thymine, cytosine, 4-N-benzoylcytosine,5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine,6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. In certainembodiments, the cleavable moiety is 2′-deoxy nucleoside that isattached to the 3′ position of the antisense oligonucleotide by aphosphodiester linkage and is attached to the linker by a phosphodiesteror phosphorothioate linkage. In certain embodiments, the cleavablemoiety is 2′-deoxy adenosine that is attached to the 3′ position of theantisense oligonucleotide by a phosphodiester linkage and is attached tothe linker by a phosphodiester or phosphorothioate linkage. In certainembodiments, the cleavable moiety is 2′-deoxy adenosine that is attachedto the 3′ position of the antisense oligonucleotide by a phosphodiesterlinkage and is attached to the linker by a phosphodiester linkage.

In certain embodiments, the cleavable moiety is attached to the 3′position of the antisense oligonucleotide. In certain embodiments, thecleavable moiety is attached to the 5′ position of the antisenseoligonucleotide. In certain embodiments, the cleavable moiety isattached to a 2′ position of the antisense oligonucleotide. In certainembodiments, the cleavable moiety is attached to the antisenseoligonucleotide by a phosphodiester linkage. In certain embodiments, thecleavable moiety is attached to the linker by either a phosphodiester ora phosphorothioate linkage. In certain embodiments, the cleavable moietyis attached to the linker by a phosphodiester linkage. In certainembodiments, the conjugate group does not include a cleavable moiety.

In certain embodiments, the cleavable moiety is cleaved after thecomplex has been administered to an animal only after being internalizedby a targeted cell. Inside the cell the cleavable moiety is cleavedthereby releasing the active antisense oligonucleotide. While notwanting to be bound by theory it is believed that the cleavable moietyis cleaved by one or more nucleases within the cell. In certainembodiments, the one or more nucleases cleave the phosphodiester linkagebetween the cleavable moiety and the linker. In certain embodiments, thecleavable moiety has a structure selected from among the following:

wherein each of Bx, Bx₁, Bx₂, and Bx₃ is independently a heterocyclicbase moiety. In certain embodiments, the cleavable moiety has astructure selected from among the following:

iii. Certain Linkers

In certain embodiments, the conjugate groups comprise a linker. Incertain such embodiments, the linker is covalently bound to thecleavable moiety. In certain such embodiments, the linker is covalentlybound to the antisense oligonucleotide. In certain embodiments, thelinker is covalently bound to a cell-targeting moiety. In certainembodiments, the linker further comprises a covalent attachment to asolid support. In certain embodiments, the linker further comprises acovalent attachment to a protein binding moiety. In certain embodiments,the linker further comprises a covalent attachment to a solid supportand further comprises a covalent attachment to a protein binding moiety.In certain embodiments, the linker includes multiple positions forattachment of tethered ligands. In certain embodiments, the linkerincludes multiple positions for attachment of tethered ligands and isnot attached to a branching group. In certain embodiments, the linkerfurther comprises one or more cleavable bond. In certain embodiments,the conjugate group does not include a linker.

In certain embodiments, the linker includes at least a linear groupcomprising groups selected from alkyl, amide, disulfide, polyethyleneglycol, ether, thioether (—S—) and hydroxylamino (—O—N(H)—) groups. Incertain embodiments, the linear group comprises groups selected fromalkyl, amide and ether groups. In certain embodiments, the linear groupcomprises groups selected from alkyl and ether groups. In certainembodiments, the linear group comprises at least one phosphorus linkinggroup. In certain embodiments, the linear group comprises at least onephosphodiester group. In certain embodiments, the linear group includesat least one neutral linking group. In certain embodiments, the lineargroup is covalently attached to the cell-targeting moiety and thecleavable moiety. In certain embodiments, the linear group is covalentlyattached to the cell-targeting moiety and the antisense oligonucleotide.In certain embodiments, the linear group is covalently attached to thecell-targeting moiety, the cleavable moiety and a solid support. Incertain embodiments, the linear group is covalently attached to thecell-targeting moiety, the cleavable moiety, a solid support and aprotein binding moiety. In certain embodiments, the linear groupincludes one or more cleavable bond.

In certain embodiments, the linker includes the linear group covalentlyattached to a scaffold group. In certain embodiments, the scaffoldincludes a branched aliphatic group comprising groups selected fromalkyl, amide, disulfide, polyethylene glycol, ether, thioether andhydroxylamino groups. In certain embodiments, the scaffold includes abranched aliphatic group comprising groups selected from alkyl, amideand ether groups. In certain embodiments, the scaffold includes at leastone mono or polycyclic ring system. In certain embodiments, the scaffoldincludes at least two mono or polycyclic ring systems. In certainembodiments, the linear group is covalently attached to the scaffoldgroup and the scaffold group is covalently attached to the cleavablemoiety and the linker. In certain embodiments, the linear group iscovalently attached to the scaffold group and the scaffold group iscovalently attached to the cleavable moiety, the linker and a solidsupport. In certain embodiments, the linear group is covalently attachedto the scaffold group and the scaffold group is covalently attached tothe cleavable moiety, the linker and a protein binding moiety. Incertain embodiments, the linear group is covalently attached to thescaffold group and the scaffold group is covalently attached to thecleavable moiety, the linker, a protein binding moiety and a solidsupport. In certain embodiments, the scaffold group includes one or morecleavable bond.

In certain embodiments, the linker includes a protein binding moiety. Incertain embodiments, the protein binding moiety is a lipid such as forexample including but not limited to cholesterol, cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A,vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g.,monosaccharide, disaccharide, trisaccharide, tetrasaccharide,oligosaccharide, polysaccharide), an endosomolytic component, a steroid(e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g.,sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid),or a cationic lipid. In certain embodiments, the protein binding moietyis a C16 to C22 long chain saturated or unsaturated fatty acid,cholesterol, cholic acid, vitamin E, adamantane or 1-pentafluoropropyl.

In certain embodiments, a linker has a structure selected from among:

wherein each n is, independently, from 1 to 20; and p is from 1 to 6.

In certain embodiments, a linker has a structure selected from among:

wherein each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

wherein each L is, independently, a phosphorus linking group or aneutral linking group; and

each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, the conjugate linker has the structure:

In certain embodiments, the conjugate linker has the structure:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

iv. Certain Cell-Targeting Moieties

In certain embodiments, conjugate groups comprise cell-targetingmoieties. Certain such cell-targeting moieties increase cellular uptakeof antisense compounds. In certain embodiments, cell-targeting moietiescomprise a branching group, one or more tether, and one or more ligand.In certain embodiments, cell-targeting moieties comprise a branchinggroup, one or more tether, one or more ligand and one or more cleavablebond.

1. Certain Branching Groups

In certain embodiments, the conjugate groups comprise a targeting moietycomprising a branching group and at least two tethered ligands. Incertain embodiments, the branching group attaches the conjugate linker.In certain embodiments, the branching group attaches the cleavablemoiety. In certain embodiments, the branching group attaches theantisense oligonucleotide. In certain embodiments, the branching groupis covalently attached to the linker and each of the tethered ligands.In certain embodiments, the branching group comprises a branchedaliphatic group comprising groups selected from alkyl, amide, disulfide,polyethylene glycol, ether, thioether and hydroxylamino groups. Incertain embodiments, the branching group comprises groups selected fromalkyl, amide and ether groups. In certain embodiments, the branchinggroup comprises groups selected from alkyl and ether groups. In certainembodiments, the branching group comprises a mono or polycyclic ringsystem. In certain embodiments, the branching group comprises one ormore cleavable bond. In certain embodiments, the conjugate group doesnot include a branching group.

In certain embodiments, a branching group has a structure selected fromamong:

wherein each n is, independently, from 1 to 20;

j is from 1 to 3; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected fromamong:

wherein each n is, independently, from 1 to 20; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected fromamong:

In certain embodiments, a branching group has a structure selected fromamong:

-   -   wherein each A₁ is independently, O, S, C═O or NH; and    -   each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected fromamong:

-   -   wherein each A₁ is independently, O, S, C═O or NH; and    -   each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected fromamong:

-   -   wherein A₁ is O, S, C═O or NH; and    -   each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected fromamong:

In certain embodiments, a branching group has a structure selected fromamong:

In certain embodiments, a branching group has a structure selected fromamong:

2. Certain Tethers

In certain embodiments, conjugate groups comprise one or more tetherscovalently attached to the branching group. In certain embodiments,conjugate groups comprise one or more tethers covalently attached to thelinking group. In certain embodiments, each tether is a linear aliphaticgroup comprising one or more groups selected from alkyl, ether,thioether, disulfide, amide and polyethylene glycol groups in anycombination. In certain embodiments, each tether is a linear aliphaticgroup comprising one or more groups selected from alkyl, substitutedalkyl, ether, thioether, disulfide, amide, phosphodiester andpolyethylene glycol groups in any combination. In certain embodiments,each tether is a linear aliphatic group comprising one or more groupsselected from alkyl, ether and amide groups in any combination. Incertain embodiments, each tether is a linear aliphatic group comprisingone or more groups selected from alkyl, substituted alkyl,phosphodiester, ether and amide groups in any combination. In certainembodiments, each tether is a linear aliphatic group comprising one ormore groups selected from alkyl and phosphodiester in any combination.In certain embodiments, each tether comprises at least one phosphoruslinking group or neutral linking group.

In certain embodiments, the tether includes one or more cleavable bond.In certain embodiments, the tether is attached to the branching groupthrough either an amide or an ether group. In certain embodiments, thetether is attached to the branching group through a phosphodiestergroup. In certain embodiments, the tether is attached to the branchinggroup through a phosphorus linking group or neutral linking group. Incertain embodiments, the tether is attached to the branching groupthrough an ether group. In certain embodiments, the tether is attachedto the ligand through either an amide or an ether group. In certainembodiments, the tether is attached to the ligand through an ethergroup. In certain embodiments, the tether is attached to the ligandthrough either an amide or an ether group. In certain embodiments, thetether is attached to the ligand through an ether group.

In certain embodiments, each tether comprises from about 8 to about 20atoms in chain length between the ligand and the branching group. Incertain embodiments, each tether group comprises from about 10 to about18 atoms in chain length between the ligand and the branching group. Incertain embodiments, each tether group comprises about 13 atoms in chainlength.

In certain embodiments, a tether has a structure selected from among:

wherein each n is, independently, from 1 to 20; and

each p is from 1 to about 6.

In certain embodiments, a tether has a structure selected from among:

In certain embodiments, a tether has a structure selected from among:

-   -   wherein each n is, independently, from 1 to 20.

In certain embodiments, a tether has a structure selected from among:

-   -   wherein L is either a phosphorus linking group or a neutral        linking group;    -   Z₁ is C(═O)O—R₂;    -   Z₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky;    -   R₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky; and    -   each m₁ is, independently, from 0 to 20 wherein at least one m₁        is greater than 0 for each tether.

In certain embodiments, a tether has a structure selected from among:

In certain embodiments, a tether has a structure selected from among:

-   -   wherein Z₂ is H or CH₃; and    -   each m₁ is, independently, from 0 to 20 wherein at least one m₁        is greater than 0 for each tether.

In certain embodiments, a tether has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, a tether comprises a phosphorus linking group.In certain embodiments, a tether does not comprise any amide bonds. Incertain embodiments, a tether comprises a phosphorus linking group anddoes not comprise any amide bonds.

3. Certain Ligands

In certain embodiments, the present disclosure provides ligands whereineach ligand is covalently attached to a tether. In certain embodiments,each ligand is selected to have an affinity for at least one type ofreceptor on a target cell. In certain embodiments, ligands are selectedthat have an affinity for at least one type of receptor on the surfaceof a mammalian liver cell. In certain embodiments, ligands are selectedthat have an affinity for the hepatic asialoglycoprotein receptor(ASGP-R). In certain embodiments, each ligand is a carbohydrate. Incertain embodiments, each ligand is, independently selected fromgalactose, N-acetyl galactoseamine, mannose, glucose, glucosamone andfucose. In certain embodiments, each ligand is N-acetyl galactoseamine(GalNAc). In certain embodiments, the targeting moiety comprises 2 to 6ligands. In certain embodiments, the targeting moiety comprises 3ligands. In certain embodiments, the targeting moiety comprises 3N-acetyl galactoseamine ligands.

In certain embodiments, the ligand is a carbohydrate, carbohydratederivative, modified carbohydrate, multivalent carbohydrate cluster,polysaccharide, modified polysaccharide, or polysaccharide derivative.In certain embodiments, the ligand is an amino sugar or a thio sugar.For example, amino sugars may be selected from any number of compoundsknown in the art, for example glucosamine, sialic acid,α-D-galactosamine, N-Acetylgalactosamine,2-acetamido-2-deoxy-D-galactopyranose (GalNAc),2-Amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose (β-muramicacid), 2-Deoxy-2-methylamino-L-glucopyranose,4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,2-Deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, andN-Glycoloyl-α-neuraminic acid. For example, thio sugars may be selectedfrom the group consisting of 5-Thio-β-D-glucopyranose, Methyl2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside,4-Thio-β-D-galactopyranose, and ethyl3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

In certain embodiments, “GalNac” or “Gal-NAc” refers to2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in theliterature as N-acetyl galactosamine. In certain embodiments, “N-acetylgalactosamine” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose. Incertain embodiments, “GalNac” or “Gal-NAc” refers to2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments,“GalNac” or “Gal-NAc” refers to2-(Acetylamino)-2-deoxy-D-galactopyranose, which includes both theβ-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form:2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, boththe β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form:2-(Acetylamino)-2-deoxy-D-galactopyranose may be used interchangeably.Accordingly, in structures in which one form is depicted, thesestructures are intended to include the other form as well. For example,where the structure for an α-form:2-(Acetylamino)-2-deoxy-D-galactopyranose is shown, this structure isintended to include the other form as well. In certain embodiments, Incertain preferred embodiments, the β-form2-(Acetylamino)-2-deoxy-D-galactopyranose is the preferred embodiment.

In certain embodiments one or more ligand has a structure selected fromamong:

wherein each R₁ is selected from OH and NHCOOH.

In certain embodiments one or more ligand has a structure selected fromamong:

In certain embodiments one or more ligand has a structure selected fromamong:

In certain embodiments one or more ligand has a structure selected fromamong:

i. Certain Conjugates

In certain embodiments, conjugate groups comprise the structuralfeatures above. In certain such embodiments, conjugate groups have thefollowing structure:

wherein each n is, independently, from 1 to 20.

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

wherein each n is, independently, from 1 to 20;

Z is H or a linked solid support;

Q is an antisense compound;

X is O or S; and

Bx is a heterocyclic base moiety.

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain embodiments, conjugates do not comprise a pyrrolidine.

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of six to elevenconsecutively bonded atoms.

In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of ten consecutivelybonded atoms.

In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of four to elevenconsecutively bonded atoms and wherein the tether comprises exactly oneamide bond.

In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted orunsubstituted alkyl, alkenyl, or alkynyl group, or a group comprising anether, a ketone, an amide, an ester, a carbamate, an amine, apiperidine, a phosphate, a phosphodiester, a phosphorothioate, atriazole, a pyrrolidine, a disulfide, or a thioether.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted orunsubstituted alkyl group, or a group comprising exactly one ether orexactly two ethers, an amide, an amine, a piperidine, a phosphate, aphosphodiester, or a phosphorothioate.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted orunsubstituted alkyl group.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein m and n are independently selected from 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, and 12.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein m is 4, 5, 6, 7, or 8, and n is 1, 2, 3, or 4.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteenconsecutively bonded atoms, and wherein X does not comprise an ethergroup.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of eightconsecutively bonded atoms, and wherein X does not comprise an ethergroup.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteenconsecutively bonded atoms, and wherein the tether comprises exactly oneamide bond, and wherein X does not comprise an ether group.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteenconsecutively bonded atoms and wherein the tether consists of an amidebond and a substituted or unsubstituted C₂-C₁₁ alkyl group.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkyl,alkenyl, or alkynyl group, or a group comprising an ether, a ketone, anamide, an ester, a carbamate, an amine, a piperidine, a phosphate, aphosphodiester, a phosphorothioate, a triazole, a pyrrolidine, adisulfide, or a thioether.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkylgroup, or a group comprising an ether, an amine, a piperidine, aphosphate, a phosphodiester, or a phosphorothioate.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkylgroup.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

Wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein n is 4, 5, 6, 7, or 8.

b. Certain Conjugated Antisense Compounds

In certain embodiments, the conjugates are bound to a nucleoside of theantisense oligonucleotide at the 2′, 3′, of 5′ position of thenucleoside. In certain embodiments, a conjugated antisense compound hasthe following structure:A-B-C-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has thefollowing structure:A-C-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least onecleavable bond.

In certain such embodiments, the branching group comprises at least onecleavable bond.

In certain embodiments each tether comprises at least one cleavablebond.

In certain embodiments, the conjugates are bound to a nucleoside of theantisense oligonucleotide at the 2′, 3′, of 5′ position of thenucleoside.

In certain embodiments, a conjugated antisense compound has thefollowing structure:A-B-C

E-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the conjugates are bound to a nucleoside of theantisense oligonucleotide at the 2′, 3′, of 5′ position of thenucleoside. In certain embodiments, a conjugated antisense compound hasthe following structure:A-C

E-F)_(q)

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has thefollowing structure:A-B-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has thefollowing structure:A-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least onecleavable bond.

In certain embodiments each tether comprises at least one cleavablebond.

In certain embodiments, a conjugated antisense compound has a structureselected from among the following:

In certain embodiments, a conjugated antisense compound has a structureselected from among the following:

In certain embodiments, a conjugated antisense compound has a structureselected from among the following:

In certain embodiments, the conjugated antisense compound has thefollowing structure:

Representative United States patents, United States patent applicationpublications, and international patent application publications thatteach the preparation of certain of the above noted conjugates,conjugated antisense compounds, tethers, linkers, branching groups,ligands, cleavable moieties as well as other modifications includewithout limitation, U.S. Pat. Nos. 5,994,517, 6,300,319, 6,660,720,6,906,182, 7,262,177, 7,491,805, 8,106,022, 7,723,509, US 2006/0148740,US 2011/0123520, WO 2013/033230 and WO 2012/037254, each of which isincorporated by reference herein in its entirety.

Representative publications that teach the preparation of certain of theabove noted conjugates, conjugated antisense compounds, tethers,linkers, branching groups, ligands, cleavable moieties as well as othermodifications include without limitation, BIESSEN et al., “TheCholesterol Derivative of a Triantennary Galactoside with High Affinityfor the Hepatic Asialoglycoprotein Receptor: a Potent CholesterolLowering Agent” J. Med. Chem. (1995) 38:1846-1852, BIESSEN et al.,“Synthesis of Cluster Galactosides with High Affinity for the HepaticAsialoglycoprotein Receptor” J. Med. Chem. (1995) 38:1538-1546, LEE etal., “New and more efficient multivalent glyco-ligands forasialoglycoprotein receptor of mammalian hepatocytes” Bioorganic &Medicinal Chemistry (2011) 19:2494-2500, RENSEN et al., “Determinationof the Upper Size Limit for Uptake and Processing of Ligands by theAsialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo” J.Biol. Chem. (2001) 276(40):37577-37584, RENSEN et al., “Design andSynthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids forTargeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor” J.Med. Chem. (2004) 47:5798-5808, SLIEDREGT et al., “Design and Synthesisof Novel Amphiphilic Dendritic Galactosides for Selective Targeting ofLiposomes to the Hepatic Asialoglycoprotein Receptor” J. Med. Chem.(1999) 42:609-618, and Valentijn et al., “Solid-phase synthesis oflysine-based cluster galactosides with high affinity for theAsialoglycoprotein Receptor” Tetrahedron, 1997, 53(2), 759-770, each ofwhich is incorporated by reference herein in its entirety.

In certain embodiments, conjugated antisense compounds comprise an RNaseH based oligonucleotide (such as a gapmer) or a splice modulatingoligonucleotide (such as a fully modified oligonucleotide) and anyconjugate group comprising at least one, two, or three GalNAc groups. Incertain embodiments a conjugated antisense compound comprises anyconjugate group found in any of the following references: Lee, CarbohydrRes, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257,939-945; Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee etal., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987,4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676;Biessen et al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al.,Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38,3487-3490; Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al.,Glycobiol, 2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276,37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlindet al., Glycoconj J, 2004, 21, 227-241; Lee et al., Bioorg Med ChemLett, 2006, 16(19), 5132-5135; Maierhofer et al., Bioorg Med Chem, 2007,15, 7661-7676; Khorev et al., Bioorg Med Chem, 2008, 16, 5216-5231; Leeet al., Bioorg Med Chem, 2011, 19, 2494-2500; Kornilova et al., AnalytBiochem, 2012, 425, 43-46; Pujol et al., Angew Chemie Int Ed Engl, 2012,51, 7445-7448; Biessen et al., J Med Chem, 1995, 38, 1846-1852;Sliedregt et al., J Med Chem, 1999, 42, 609-618; Rensen et al., J MedChem, 2004, 47, 5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol,2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464;Sato et al., J Am Chem Soc, 2004, 126, 14013-14022; Lee et al., J OrgChem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792;Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., MethodsEnzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14,18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan,Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al.,Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013,21, 5275-5281; International applications WO1998/013381; WO2011/038356;WO1997/046098; WO2008/098788; WO2004/101619; WO2012/037254;WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947;WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046;WO2009/082607; WO2009/134487; WO2010/144740; WO2010/148013;WO1997/020563; WO2010/088537; WO2002/043771; WO2010/129709;WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406;WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; U.S. Pat.Nos. 4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319;8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812;6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772;8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182;6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. PatentApplication Publications US2011/0097264; US2011/0097265; US2013/0004427;US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730;US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814;US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393;US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075;US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938;US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968;US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132; eachof which is incorporated by reference in its entirety.

C. Certain Uses and Features

In certain embodiments, conjugated antisense compounds exhibit potenttarget RNA reduction in vivo. In certain embodiments, unconjugatedantisense compounds accumulate in the kidney. In certain embodiments,conjugated antisense compounds accumulate in the liver. In certainembodiments, conjugated antisense compounds are well tolerated. Suchproperties render conjugated antisense compounds particularly useful forinhibition of many target RNAs, including, but not limited to thoseinvolved in metabolic, cardiovascular and other diseases, disorders orconditions. Thus, provided herein are methods of treating such diseases,disorders or conditions by contacting liver tissues with the conjugatedantisense compounds targeted to RNAs associated with such diseases,disorders or conditions. Thus, also provided are methods forameliorating any of a variety of metabolic, cardiovascular and otherdiseases, disorders or conditions with the conjugated antisensecompounds of the present invention.

In certain embodiments, conjugated antisense compounds are more potentthan unconjugated counterpart at a particular tissue concentration.Without wishing to be bound by any theory or mechanism, in certainembodiments, the conjugate may allow the conjugated antisense compoundto enter the cell more efficiently or to enter the cell moreproductively. For example, in certain embodiments conjugated antisensecompounds may exhibit greater target reduction as compared to itsunconjugated counterpart wherein both the conjugated antisense compoundand its unconjugated counterpart are present in the tissue at the sameconcentrations. For example, in certain embodiments conjugated antisensecompounds may exhibit greater target reduction as compared to itsunconjugated counterpart wherein both the conjugated antisense compoundand its unconjugated counterpart are present in the liver at the sameconcentrations.

Productive and non-productive uptake of oligonucleotides has beendiscussed previously (See e.g. Geary, R. S., E. Wancewicz, et al.(2009). “Effect of Dose and Plasma Concentration on Liver Uptake andPharmacologic Activity of a 2′-Methoxyethyl Modified Chimeric AntisenseOligonucleotide Targeting PTEN.” Biochem. Pharmacol. 78(3): 284-91; &Koller, E., T. M. Vincent, et al. (2011). “Mechanisms of single-strandedphosphorothioate modified antisense oligonucleotide accumulation inhepatocytes.” Nucleic Acids Res. 39(11): 4795-807). Conjugate groupsdescribed herein may improve productive uptake.

In certain embodiments, the conjugate groups described herein mayfurther improve potency by increasing the affinity of the conjugatedantisense compound for a particular type of cell or tissue. In certainembodiments, the conjugate groups described herein may further improvepotency by increasing recognition of the conjugated antisense compoundby one or more cell-surface receptors. In certain embodiments, theconjugate groups described herein may further improve potency byfacilitating endocytosis of the conjugated antisense compound.

In certain embodiments, the cleavable moiety may further improve potencyby allowing the conjugate to be cleaved from the antisenseoligonucleotide after the conjugated antisense compound has entered thecell. Accordingly, in certain embodiments, conjugated antisensecompounds can be administer at doses lower than would be necessary forunconjugated antisense oligonucleotides.

Phosphorothioate linkages have been incorporated into antisenseoligonucleotides previously. Such phosphorothioate linkages areresistant to nucleases and so improve stability of the oligonucleotide.Further, phosphorothioate linkages also bind certain proteins, whichresults in accumulation of antisense oligonucleotide in the liver.Oligonucleotides with fewer phosphorothioate linkages accumulate less inthe liver and more in the kidney (see, for example, Geary, R.,“Pharmacokinetic Properties of 2′-O-(2-Methoxyethyl)-ModifiedOligonucleotide Analogs in Rats,” Journal of Pharmacology andExperimental Therapeutics, Vol. 296, No. 3, 890-897; & PharmacologicalProperties of 2′-O-Methoxyethyl Modified Oligonucleotides in Antisense aDrug Technology, Chapter 10, Crooke, S. T., ed., 2008) In certainembodiments, oligonucleotides with fewer phosphorothioateinternculeoside linkages and more phosphodiester internucleosidelinkages accumulate less in the liver and more in the kidney. Whentreating diseases in the liver, this is undesirable for several reasons(1) less drug is getting to the site of desired action (liver); (2) drugis escaping into the urine; and (3) the kidney is exposed to relativelyhigh concentration of drug which can result in toxicities in the kidney.Thus, for liver diseases, phosphorothioate linkages provide importantbenefits.

In certain embodiments, however, administration of oligonucleotidesuniformly linked by phosphorothioate internucleoside linkages inducesone or more proinflammatory reactions. (see for example: J Lab Clin Med.1996 September; 128(3):329-38. “Amplification of antibody production byphosphorothioate oligodeoxynucleotides”. Branda et al.; and see also forexample: Toxicologic Properties in Antisense a Drug Technology, Chapter12, pages 342-351, Crooke, S. T., ed., 2008). In certain embodiments,administration of oligonucleotides wherein most of the internucleosidelinkages comprise phosphorothioate internucleoside linkages induces oneor more proinflammatory reactions.

In certain embodiments, the degree of proinflammatory effect may dependon several variables (e.g. backbone modification, off-target effects,nucleobase modifications, and/or nucleoside modifications) see forexample: Toxicologic Properties in Antisense a Drug Technology, Chapter12, pages 342-351, Crooke, S. T., ed., 2008). In certain embodiments,the degree of proinflammatory effect may be mitigated by adjusting oneor more variables. For example the degree of proinflammatory effect of agiven oligonucleotide may be mitigated by replacing any number ofphosphorothioate internucleoside linkages with phosphodiesterinternucleoside linkages and thereby reducing the total number ofphosphorothioate internucleoside linkages.

In certain embodiments, it would be desirable to reduce the number ofphosphorothioate linkages, if doing so could be done without losingstability and without shifting the distribution from liver to kidney.For example, in certain embodiments, the number of phosphorothioatelinkages may be reduced by replacing phosphorothioate linkages withphosphodiester linkages. In such an embodiment, the antisense compoundhaving fewer phosphorothioate linkages and more phosphodiester linkagesmay induce less proinflammatory reactions or no proinflammatoryreaction. Although the antisense compound having fewer phosphorothioatelinkages and more phosphodiester linkages may induce fewerproinflammatory reactions, the antisense compound having fewerphosphorothioate linkages and more phosphodiester linkages may notaccumulate in the liver and may be less efficacious at the same orsimilar dose as compared to an antisense compound having morephosphorothioate linkages. In certain embodiments, it is thereforedesirable to design an antisense compound that has a plurality ofphosphodiester bonds and a plurality of phosphorothioate bonds but whichalso possesses stability and good distribution to the liver.

In certain embodiments, conjugated antisense compounds accumulate morein the liver and less in the kidney than unconjugated counterparts, evenwhen some of the phosporothioate linkages are replaced with lessproinflammatory phosphodiester internucleoside linkages. In certainembodiments, conjugated antisense compounds accumulate more in the liverand are not excreted as much in the urine compared to its unconjugatedcounterparts, even when some of the phosporothioate linkages arereplaced with less proinflammatory phosphodiester internucleosidelinkages. In certain embodiments, the use of a conjugate allows one todesign more potent and better tolerated antisense drugs. Indeed, incertain embodiments, conjugated antisense compounds have largertherapeutic indexes than unconjugated counterparts. This allows theconjugated antisense compound to be administered at a higher absolutedose, because there is less risk of proinflammatory response and lessrisk of kidney toxicity. This higher dose, allows one to dose lessfrequently, since the clearance (metabolism) is expected to be similar.Further, because the compound is more potent, as described above, onecan allow the concentration to go lower before the next dose withoutlosing therapeutic activity, allowing for even longer periods betweendosing.

In certain embodiments, the inclusion of some phosphorothioate linkagesremains desirable. For example, the terminal linkages are vulnerable toexonucleases and so in certain embodiments, those linkages arephosphorothioate or other modified linkage. Internucleoside linkageslinking two deoxynucleosides are vulnerable to endonucleases and so incertain embodiments those linkages are phosphorothioate or othermodified linkage. Internucleoside linkages between a modified nucleosideand a deoxynucleoside where the deoxynucleoside is on the 5′ side of thelinkage deoxynucleosides are vulnerable to endonucleases and so incertain embodiments those linkages are phosphorothioate or othermodified linkage. Internucleoside linkages between two modifiednucleosides of certain types and between a deoxynucleoside and amodified nucleoside of certain type where the modified nucleoside is atthe 5′ side of the linkage are sufficiently resistant to nucleasedigestion, that the linkage can be phosphodiester.

In certain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 16 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 15 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 14 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 13 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 12 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 11 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 10 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 9 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 8 phosphorthioate linkages.

In certain embodiments, antisense compounds comprising one or moreconjugate group described herein has increased activity and/or potencyand/or tolerability compared to a parent antisense compound lacking suchone or more conjugate group. Accordingly, in certain embodiments,attachment of such conjugate groups to an oligonucleotide is desirable.Such conjugate groups may be attached at the 5′-, and/or 3′-end of anoligonucleotide. In certain instances, attachment at the 5′-end issynthetically desirable. Typically, oligonuclotides are synthesized byattachment of the 3′ terminal nucleoside to a solid support andsequential coupling of nucleosides from 3′ to 5′ using techniques thatare well known in the art. Accordingly if a conjugate group is desiredat the 3′-terminus, one may (1) attach the conjugate group to the3′-terminal nucleoside and attach that conjugated nucleoside to thesolid support for subsequent preparation of the oligonucleotide or (2)attach the conjugate group to the 3′-terminal nucleoside of a completedoligonucleotide after synthesis. Neither of these approaches is veryefficient and thus both are costly. In particular, attachment of theconjugated nucleoside to the solid support, while demonstrated in theExamples herein, is an inefficient process. In certain embodiments,attaching a conjugate group to the 5′-terminal nucleoside issynthetically easier than attachment at the 3′-end. One may attach anon-conjugated 3′ terminal nucleoside to the solid support and preparethe oligonucleotide using standard and well characterized reactions. Onethen needs only to attach a 5′ nucleoside having a conjugate group atthe final coupling step. In certain embodiments, this is more efficientthan attaching a conjugated nucleoside directly to the solid support asis typically done to prepare a 3′-conjugated oligonucleotide. TheExamples herein demonstrate attachment at the 5′-end. In addition,certain conjugate groups have synthetic advantages. For Example, certainconjugate groups comprising phosphorus linkage groups are syntheticallysimpler and more efficiently prepared than other conjugate groups,including conjugate groups reported previously (e.g., WO/2012/037254).

In certain embodiments, conjugated antisense compounds are administeredto a subject. In such embodiments, antisense compounds comprising one ormore conjugate group described herein has increased activity and/orpotency and/or tolerability compared to a parent antisense compoundlacking such one or more conjugate group. Without being bound bymechanism, it is believed that the conjugate group helps withdistribution, delivery, and/or uptake into a target cell or tissue. Incertain embodiments, once inside the target cell or tissue, it isdesirable that all or part of the conjugate group to be cleaved torelease the active oligonucleotide. In certain embodiments, it is notnecessary that the entire conjugate group be cleaved from theoligonucleotide. For example, in Example 20 a conjugated oligonucleotidewas administered to mice and a number of different chemical species,each comprising a different portion of the conjugate group remaining onthe oligonucleotide, were detected (Table 23a). This conjugatedantisense compound demonstrated good potency (Table 23). Thus, incertain embodiments, such metabolite profile of multiple partialcleavage of the conjugate group does not interfere withactivity/potency. Nevertheless, in certain embodiments it is desirablethat a prodrug (conjugated oligonucleotide) yield a single activecompound. In certain instances, if multiple forms of the active compoundare found, it may be necessary to determine relative amounts andactivities for each one. In certain embodiments where regulatory reviewis required (e.g., USFDA or counterpart) it is desirable to have asingle (or predominantly single) active species. In certain suchembodiments, it is desirable that such single active species be theantisense oligonucleotide lacking any portion of the conjugate group. Incertain embodiments, conjugate groups at the 5′-end are more likely toresult in complete metabolism of the conjugate group. Without beingbound by mechanism it may be that endogenous enzymes responsible formetabolism at the 5′ end (e.g., 5′ nucleases) are more active/efficientthan the 3′ counterparts. In certain embodiments, the specific conjugategroups are more amenable to metabolism to a single active species. Incertain embodiments, certain conjugate groups are more amenable tometabolism to the oligonucleotide.

D. Antisense

In certain embodiments, oligomeric compounds of the present inventionare antisense compounds. In such embodiments, the oligomeric compound iscomplementary to a target nucleic acid. In certain embodiments, a targetnucleic acid is an RNA. In certain embodiments, a target nucleic acid isa non-coding RNA. In certain embodiments, a target nucleic acid encodesa protein. In certain embodiments, a target nucleic acid is selectedfrom a mRNA, a pre-mRNA, a microRNA, a non-coding RNA, including smallnon-coding RNA, and a promoter-directed RNA. In certain embodiments,oligomeric compounds are at least partially complementary to more thanone target nucleic acid. For example, oligomeric compounds of thepresent invention may be microRNA mimics, which typically bind tomultiple targets.

In certain embodiments, antisense compounds comprise a portion having anucleobase sequence at least 70% complementary to the nucleobasesequence of a target nucleic acid. In certain embodiments, antisensecompounds comprise a portion having a nucleobase sequence at least 80%complementary to the nucleobase sequence of a target nucleic acid. Incertain embodiments, antisense compounds comprise a portion having anucleobase sequence at least 90% complementary to the nucleobasesequence of a target nucleic acid. In certain embodiments, antisensecompounds comprise a portion having a nucleobase sequence at least 95%complementary to the nucleobase sequence of a target nucleic acid. Incertain embodiments, antisense compounds comprise a portion having anucleobase sequence at least 98% complementary to the nucleobasesequence of a target nucleic acid. In certain embodiments, antisensecompounds comprise a portion having a nucleobase sequence that is 100%complementary to the nucleobase sequence of a target nucleic acid. Incertain embodiments, antisense compounds are at least 70%, 80%, 90%,95%, 98%, or 100% complementary to the nucleobase sequence of a targetnucleic acid over the entire length of the antisense compound.

Antisense mechanisms include any mechanism involving the hybridizationof an oligomeric compound with target nucleic acid, wherein thehybridization results in a biological effect. In certain embodiments,such hybridization results in either target nucleic acid degradation oroccupancy with concomitant inhibition or stimulation of the cellularmachinery involving, for example, translation, transcription, orpolyadenylation of the target nucleic acid or of a nucleic acid withwhich the target nucleic acid may otherwise interact.

One type of antisense mechanism involving degradation of target RNA isRNase H mediated antisense. RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. It is known in the art thatsingle-stranded antisense compounds which are “DNA-like” elicit RNase Hactivity in mammalian cells. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof DNA-like oligonucleotide-mediated inhibition of gene expression.

Antisense mechanisms also include, without limitation RNAi mechanisms,which utilize the RISC pathway. Such RNAi mechanisms include, withoutlimitation siRNA, ssRNA and microRNA mechanisms. Such mechanisms includecreation of a microRNA mimic and/or an anti-microRNA.

Antisense mechanisms also include, without limitation, mechanisms thathybridize or mimic non-coding RNA other than microRNA or mRNA. Suchnon-coding RNA includes, but is not limited to promoter-directed RNA andshort and long RNA that effects transcription or translation of one ormore nucleic acids.

In certain embodiments, oligonucleotides comprising conjugates describedherein are RNAi compounds. In certain embodiments, oligomericoligonucleotides comprising conjugates described herein are ssRNAcompounds. In certain embodiments, oligonucleotides comprisingconjugates described herein are paired with a second oligomeric compoundto form an siRNA. In certain such embodiments, the second oligomericcompound also comprises a conjugate. In certain embodiments, the secondoligomeric compound is any modified or unmodified nucleic acid. Incertain embodiments, the oligonucleotides comprising conjugatesdescribed herein is the antisense strand in an siRNA compound. Incertain embodiments, the oligonucleotides comprising conjugatesdescribed herein is the sense strand in an siRNA compound. Inembodiments in which the conjugated oligomeric compound isdouble-stranded siRnA, the conjugate may be on the sense strand, theantisense strand or both the sense strand and the antisense strand.

C. Apolipoprotein (a) (Apo(a))

In certain embodiments, conjugated antisense compounds target any apo(a)nucleic acid. In certain embodiments, the target nucleic acid encodes anapo(a) target protein that is clinically relevant. In such embodiments,modulation of the target nucleic acid results in clinical benefit.

The targeting process usually includes determination of at least onetarget region, segment, or site within the target nucleic acid for theantisense interaction to occur such that the desired effect will result.

In certain embodiments, a target region is a structurally defined regionof the nucleic acid. For example, in certain such embodiments, a targetregion may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region or target segment.

In certain embodiments, a target segment is at least about an8-nucleobase portion of a target region to which a conjugated antisensecompound is targeted. Target segments can include DNA or RNA sequencesthat comprise at least 8 consecutive nucleobases from the 5′-terminus ofone of the target segments (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA comprises about 8 to about 30 nucleobases). Targetsegments are also represented by DNA or RNA sequences that comprise atleast 8 consecutive nucleobases from the 3′-terminus of one of thetarget segments (the remaining nucleobases being a consecutive stretchof the same DNA or RNA beginning immediately downstream of the3′-terminus of the target segment and continuing until the DNA or RNAcomprises about 8 to about 30 nucleobases). Target segments can also berepresented by DNA or RNA sequences that comprise at least 8 consecutivenucleobases from an internal portion of the sequence of a targetsegment, and may extend in either or both directions until theconjugated antisense compound comprises about 8 to about 30 nucleobases.

In certain embodiments, antisense compounds targeted to an apo(a)nucleic acid can be modified as described herein. In certainembodiments, the antisense compounds can have a modified sugar moiety,an unmodified sugar moiety or a mixture of modified and unmodified sugarmoieties as described herein. In certain embodiments, the antisensecompounds can have a modified internucleoside linkage, an unmodifiedinternucleoside linkage or a mixture of modified and unmodifiedinternucleoside linkages as described herein. In certain embodiments,the antisense compounds can have a modified nucleobase, an unmodifiednucleobase or a mixture of modified and unmodified nucleobases asdescribed herein. In certain embodiments, the antisense compounds canhave a motif as described herein.

In certain embodiments, antisense compounds targeted to apo(a) nucleicacids can be conjugated as described herein.

One apo(a) protein is linked via a disulfide bond to a singleapolipoprotein B (apoB) protein to form a lipoprotein(a) (Lp(a))particle. The apo(a) protein shares a high degree of homology withplasminogen particularly within the kringle IV type 2 repetitive domain.It is thought that the kringle repeat domain in apo(a) may beresponsible for its pro-thrombotic and anti-fibrinolytic properties,potentially enhancing atherosclerotic progression. Apo(a) istranscriptionally regulated by IL-6 and in studies in rheumatoidarthritis patients treated with an IL-6 inhibitor (tocilizumab), plasmalevels were reduced by 30% after 3 month treatment. Apo(a) has beenshown to preferentially bind oxidized phospholipids and potentiatevascular inflammation. Further, studies suggest that the Lp(a) particlemay also stimulate endothelial permeability, induce plasminogenactivator inhibitor type-1 expression and activate macrophageinterleukin-8 secretion. Importantly, recent genetic association studiesrevealed that Lp(a) was an independent risk factor for myocardialinfarction, stroke, peripheral vascular disease and abdominal aorticaneurysm. Further, in the Precocious Coronary Artery Disease (PROCARDIS)study, Clarke et al. described robust and independent associationsbetween coronary heart disease and plasma Lp(a) concentrations.Additionally, Solfrizzi et al., suggested that increased serum Lp(a) maybe linked to an increased risk for Alzheimer's Disease (AD). Antisensecompounds targeting apo(a) have been previously disclosed inWO2005/000201 and US2010-0331390, herein incorporated by reference inits entirety. An antisense oligonucleobase targeting Apo(a),ISIS-APOA_(Rx), was assessed in a Phase I clinical trial to study it'ssafety profile.

Certain Conjugated Antisense Compounds Targeted to an Apo(a) NucleicAcid

In certain embodiments, conjugated antisense compounds are targeted toan Apo(a) nucleic acid having the sequence of GENBANK® Accession No.NM_005577.2, incorporated herein as SEQ ID NO: 1; GENBANK Accession No.NT_007422.12 truncated from nucleotides 3230000 to 3380000, incorporatedherein as SEQ ID NO: 2; GENBANK Accession No. NT_025741.15 truncatedfrom nucleotides 65120000 to 65258000, designated herein as SEQ ID NO:3; and GENBANK Accession No. NM_005577.1, incorporated herein as SEQ IDNO: 4. In certain such embodiments, a conjugated antisense compound isat least 90%, at least 95%, or 100% complementary to any of thenucleobase sequences of SEQ ID NOs: 1-4.

In certain embodiments, a conjugated antisense compound targeted to anyof the nucleobase sequences of SEQ ID NOs: 1-4 comprises an at least 8consecutive nucleobase sequence selected from the nucleobase sequence ofany of SEQ ID NOs: 12-130, 133, 134. In certain embodiments, aconjugated antisense compound targeted to any of SEQ ID NOs: 1-4comprises a nucleobase sequence selected from the nucleobase sequence ofany of SEQ ID NOs: 12-130, 133, 134.

TABLE A Antisense Compounds targeted to Apo(a) SEQ ID NO: 1 Target StartSEQ ID ISIS No Site Sequence (5′-3′) Motif NO 494372 3901TGCTCCGTTGGTGCTTGTTC eeeeeddddddddddeeeee 58 494283 584TCTTCCTGTGACAGTGGTGG eeeeeddddddddddeeeee 26 926 1610 1952 2294 3320494284 585 TTCTTCCTGTGACAGTGGTG eeeeeddddddddddeeeee 27 927 1611 19532295 3321 494286 587 GGTTCTTCCTGTGACAGTGG eeeeeddddddddddeeeee 29 9291613 1955 2297 494301 628 CGACTATGCGAGTGTGGTGT eeeeeddddddddddeeeee 38970 1312 1654 1996 2338 2680 3022 494302 629 CCGACTATGCGAGTGTGGTGeeeeeddddddddddeeeee 39 971 1313 1655 1997 2339 2681 3023Apo(a) Therapeutic Indications

In certain embodiments, the invention provides methods for using aconjugated antisense compound targeted to an apo(a) nucleic acid formodulating the expression of apo(a) in a subject. In certainembodiments, the expression of apo(a) is reduced.

In certain embodiments, provided herein are methods of treating asubject comprising administering one or more pharmaceutical compositionsas described herein. In certain embodiments, the invention providesmethods for using a conjugated antisense compound targeted to an apo(a)nucleic acid in a pharmaceutical composition for treating a subject. Incertain embodiments, the individual has an apo(a) related disease. Incertain embodiments, the individual has an Lp(a) related disease. Incertain embodiments, the individual has an inflammatory, cardiovascularand/or a metabolic disease, disorder or condition.

In certain embodiments, the subject has an inflammatory, cardiovascularand/or metabolic disease, disorder or condition.

In certain embodiments, the cardiovascular diseases, disorders orconditions include, but are not limited to, aortic stenosis, aneurysm(e.g., abdominal aortic aneurysm), angina, arrhythmia, atherosclerosis,cerebrovascular disease, coronary artery disease, coronary heartdisease, dyslipidemia, hypercholesterolemia, hyperlipidemia,hypertension, hypertriglyceridemia, myocardial infarction, peripheralvascular disease (e.g., peripheral artery disease), stroke and the like.

In certain embodiments, the compounds targeted to apo(a) describedherein modulate physiological markers or phenotypes of thecardiovascular disease, disorder or condition. For example,administration of the compounds to animals can decrease LDL andcholesterol levels in those animals compared to untreated animals. Incertain embodiments, the modulation of the physiological markers orphenotypes can be associated with inhibition of apo(a) by the compounds.

In certain embodiments, the physiological markers of the cardiovasculardisease, disorder or condition can be quantifiable. For example, LDL orcholesterol levels can be measured and quantified by, for example,standard lipid tests. For such markers, in certain embodiments, themarker can be decreased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any twoof these values.

Also, provided herein are methods for preventing, treating orameliorating a symptom associated with the cardiovascular disease,disorder or condition in a subject in need thereof. In certainembodiments, provided is a method for reducing the rate of onset of asymptom associated with the cardiovascular disease, disorder orcondition. In certain embodiments, provided is a method for reducing theseverity of a symptom associated with the cardiovascular disease,disorder or condition. In such embodiments, the methods compriseadministering a therapeutically effective amount of a compound targetedto an apo(a) nucleic acid to an individual in need thereof.

The cardiovascular disease, disorder or condition can be characterizedby numerous physical symptoms. Any symptom known to one of skill in theart to be associated with the cardiovascular disease, disorder orcondition can be prevented, treated, ameliorated or otherwise modulatedwith the compounds and methods described herein. In certain embodiments,the symptom can be any of, but not limited to, angina, chest pain,shortness of breath, palpitations, weakness, dizziness, nausea,sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation,swelling in the lower extremities, cyanosis, fatigue, fainting, numbnessof the face, numbness of the limbs, claudication or cramping of muscles,bloating of the abdomen or fever.

In certain embodiments, the metabolic diseases, disorders or conditionsinclude, but are not limited to, hyperglycemia, prediabetes, diabetes(type I and type II), obesity, insulin resistance, metabolic syndromeand diabetic dyslipidemia.

In certain embodiments, compounds targeted to apo(a) as described hereinmodulate physiological markers or phenotypes of the metabolic disease,disorder or condition. For example, administration of the compounds toanimals can decrease glucose and insulin resistance levels in thoseanimals compared to untreated animals. In certain embodiments, themodulation of the physiological markers or phenotypes can be associatedwith inhibition of apo(a) by the compounds.

In certain embodiments, physiological markers of the metabolic disease,disorder or condition can be quantifiable. For example, glucose levelsor insulin resistance can be measured and quantified by standard testsknown in the art. For such markers, in certain embodiments, the markercan be decreased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two ofthese values. In another example, insulin sensitivity can be measuredand quantified by standard tests known in the art. For such markers, incertain embodiments, the marker can be increase by about 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or arange defined by any two of these values.

Also, provided herein are methods for preventing, treating orameliorating a symptom associated with the metabolic disease, disorderor condition in a subject in need thereof. In certain embodiments,provided is a method for reducing the rate of onset of a symptomassociated with the metabolic disease, disorder or condition. In certainembodiments, provided is a method for reducing the severity of a symptomassociated with the metabolic disease, disorder or condition. In suchembodiments, the methods comprise administering a therapeuticallyeffective amount of a compound targeted to an apo(a) nucleic acid to anindividual in need thereof.

The metabolic disease, disorder or condition can be characterized bynumerous physical symptoms. Any symptom known to one of skill in the artto be associated with the metabolic disease, disorder or condition canbe prevented, treated, ameliorated or otherwise modulated with thecompounds and methods described herein. In certain embodiments, thesymptom can be any of, but not limited to, excessive urine production(polyuria), excessive thirst and increased fluid intake (polydipsia),blurred vision, unexplained weight loss and lethargy.

In certain embodiments, the inflammatory diseases, disorders orconditions include, but are not limited to, aortic stenosis, coronaryartey disease (CAD), Alzheimer's Disease and thromboembolic diseases,disorder or conditions. Certain thromboembolic diseases, disorders orconditions include, but are not limited to, stroke, thrombosis,myocardial infarction and peripheral vascular disease.

In certain embodiments, the compounds targeted to apo(a) describedherein modulate physiological markers or phenotypes of the inflammatorydisease, disorder or condition. For example, administration of thecompounds to animals can decrease inflammatory cytokine or otherinflammatory markers levels in those animals compared to untreatedanimals. In certain embodiments, the modulation of the physiologicalmarkers or phenotypes can be associated with inhibition of apo(a) by thecompounds.

In certain embodiments, the physiological markers of the inflammatorydisease, disorder or condition can be quantifiable. For example,cytokine levels can be measured and quantified by standard tests knownin the art. For such markers, in certain embodiments, the marker can bedecreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or a rangedefined by any two of these values.

Also, provided herein are methods for preventing, treating orameliorating a symptom associated with the inflammatory disease,disorder or condition in a subject in need thereof. In certainembodiments, provided is a method for reducing the rate of onset of asymptom associated with the inflammatory disease, disorder or condition.In certain embodiments, provided is a method for reducing the severityof a symptom associated with the inflammatory disease, disorder orcondition. In such embodiments, the methods comprise administering atherapeutically effective amount of a compound targeted to an apo(a)nucleic acid to an individual in need thereof.

In certain embodiments, provided are methods of treating an individualwith an apo(a) related disease, disorder or condition comprisingadministering a therapeutically effective amount of one or morepharmaceutical compositions as described herein. In certain embodiments,the individual has elevated apo(a) levels. In certain embodiments,provided are methods of treating an individual with an Lp(a) relateddisease, disorder or condition comprising administering atherapeutically effective amount of one or more pharmaceuticalcompositions as described herein. In certain embodiments, the individualhas elevated Lp(a) levels. In certain embodiments, the individual has aninflammatory, cardiovascular and/or metabolic disease, disorder orcondition. In certain embodiments, administration of a therapeuticallyeffective amount of an antisense compound targeted to an apo(a) nucleicacid is accompanied by monitoring of apo(a) or Lp(a) levels. In certainembodiments, administration of a therapeutically effective amount of anantisense compound targeted to an apo(a) nucleic acid is accompanied bymonitoring of markers of inflammatory, cardiovascular and/or metabolicdisease, or other disease process associated with the expression ofapo(a), to determine an individual's response to the antisense compound.An individual's response to administration of the antisense compoundtargeting apo(a) can be used by a physician to determine the amount andduration of therapeutic intervention with the compound.

In certain embodiments, administration of an antisense compound targetedto an apo(a) nucleic acid results in reduction of apo(a) expression byat least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 99%, or a range defined by any two ofthese values. In certain embodiments, apo(a) expression is reduced to atleast ≤100 mg/dL, ≤90 mg/dL, ≤80 mg/dL, ≤70 mg/dL, ≤60 mg/dL, ≤50 mg/dL,≤40 mg/dL, ≤30 mg/dL, ≤20 mg/dL or ≤10 mg/dL.

In certain embodiments, administration of an antisense compound targetedto an apo(a) nucleic acid results in reduction of Lp(a) expression by atleast about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 99%, or a range defined by any two of thesevalues. In certain embodiments, Lp(a) expression is reduced to at least≤200 mg/dL, ≤190 mg/dL, ≤180 mg/dL, ≤175 mg/dL, ≤170 mg/dL, ≤160 mg/dL,≤150 mg/dL, ≤140 mg/dL, ≤130 mg/dL, ≤120 mg/dL, ≤110 mg/dL, ≤100 mg/dL,≤90 mg/dL, ≤80 mg/dL, ≤70 mg/dL, ≤60 mg/dL, ≤55 mg/dL, ≤50 mg/dL, ≤45mg/dL, ≤40 mg/dL, ≤35 mg/dL, ≤30 mg/dL, ≤25 mg/dL, ≤20 mg/dL, ≤15 mg/dL,or ≤10 mg/dL.

In certain embodiments, the invention provides methods for using aconjugated antisense compound targeted to an apo(a) nucleic acid in thepreparation of a medicament. In certain embodiments, pharmaceuticalcompositions comprising a conjugated antisense compound targeted toapo(a) are used for the preparation of a medicament for treating apatient suffering or susceptible to an inflammatory, cardiovascularand/or a metabolic disease, disorder or condition.

Apo(a) Treatment Populations

Certain subjects with high Lp(a) levels are at a significant risk ofvarious diseases (Lippi et al., Clinica Chimica Acta, 2011, 412:797-801;Solfrizz et al.). In many subjects with high Lp(a) levels, currenttreatments cannot reduce their Lp(a) levels to safe levels. Apo(a) playsan important role in the formation of Lp(a), hence reducing apo(a) canreduce Lp(a) and prevent, treat or ameliorate a disease associated withLp(a).

In certain embodiments, treatment with the compounds and methodsdisclosed herein is indicated for a human animal with elevated apo(a)levels and/or Lp(a) levels. In certain embodiments, the human has apo(a)levels ≥10 mg/dL, ≥20 mg/dL, ≥30 mg/dL, ≥40 mg/dL, ≥50 mg/dL, ≥60 mg/dL,≥70 mg/dL, ≥80 mg/dL, ≥90 mg/dL or ≥100 mg/dL. In certain embodiments,the human has Lp(a) levels ≥10 mg/dL, ≥15 mg/dL, ≥20 mg/dL, ≥25 mg/dL,≥30 mg/dL, ≥35 mg/dL, ≥40 mg/dL, ≥50 mg/dL, ≥60 mg/dL, ≥70 mg/dL, ≥80mg/dL, ≥90 mg/dL, ≥100 mg/dL, ≥110 mg/dL, ≥120 mg/dL, ≥130 mg/dL, ≥140mg/dL, ≥150 mg/dL, ≥160 mg/dL, ≥170 mg/dL, ≥175 mg/dL, ≥180 mg/dL, ≥190mg/dL, ≥200 mg/dL.

D. Certain Pharmaceutical Compositions

In certain embodiments, the present disclosure provides pharmaceuticalcompositions comprising one or more antisense compound. In certainembodiments, such pharmaceutical composition comprises a suitablepharmaceutically acceptable diluent or carrier. In certain embodiments,a pharmaceutical composition comprises a sterile saline solution and oneor more antisense compound. In certain embodiments, such pharmaceuticalcomposition consists of a sterile saline solution and one or moreantisense compound. In certain embodiments, the sterile saline ispharmaceutical grade saline. In certain embodiments, a pharmaceuticalcomposition comprises one or more antisense compound and sterile water.In certain embodiments, a pharmaceutical composition consists of one ormore antisense compound and sterile water. In certain embodiments, thesterile saline is pharmaceutical grade water. In certain embodiments, apharmaceutical composition comprises one or more antisense compound andphosphate-buffered saline (PBS). In certain embodiments, apharmaceutical composition consists of one or more antisense compoundand sterile phosphate-buffered saline (PBS). In certain embodiments, thesterile saline is pharmaceutical grade PBS.

In certain embodiments, antisense compounds may be admixed withpharmaceutically acceptable active and/or inert substances for thepreparation of pharmaceutical compositions or formulations. Compositionsand methods for the formulation of pharmaceutical compositions depend ona number of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters. Incertain embodiments, pharmaceutical compositions comprising antisensecompounds comprise one or more oligonucleotide which, uponadministration to an animal, including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of antisense compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an oligonucleotide which are cleaved by endogenousnucleases within the body, to form the active antisense oligonucleotide.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In certain such methods, the nucleic acid is introduced intopreformed liposomes or lipoplexes made of mixtures of cationic lipidsand neutral lipids. In certain methods, DNA complexes with mono- orpoly-cationic lipids are formed without the presence of a neutral lipid.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to a particular cell or tissue.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to fat tissue. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions provided hereincomprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided hereincomprises a delivery system. Examples of delivery systems include, butare not limited to, liposomes and emulsions. Certain delivery systemsare useful for preparing certain pharmaceutical compositions includingthose comprising hydrophobic compounds. In certain embodiments, certainorganic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided hereincomprises one or more tissue-specific delivery molecules designed todeliver the one or more pharmaceutical agents of the present disclosureto specific tissues or cell types. For example, in certain embodiments,pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided hereincomprises a co-solvent system. Certain of such co-solvent systemscomprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition provided herein isprepared for oral administration. In certain embodiments, pharmaceuticalcompositions are prepared for buccal administration.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared fortransmucosal administration. In certain of such embodiments penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition provided hereincomprises an oligonucleotide in a therapeutically effective amount. Incertain embodiments, the therapeutically effective amount is sufficientto prevent, alleviate or ameliorate symptoms of a disease or to prolongthe survival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In certain embodiments, one or more modified oligonucleotide providedherein is formulated as a prodrug. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically more active form of anoligonucleotide. In certain embodiments, prodrugs are useful becausethey are easier to administer than the corresponding active form. Forexample, in certain instances, a prodrug may be more bioavailable (e.g.,through oral administration) than is the corresponding active form. Incertain instances, a prodrug may have improved solubility compared tothe corresponding active form. In certain embodiments, prodrugs are lesswater soluble than the corresponding active form. In certain instances,such prodrugs possess superior transmittal across cell membranes, wherewater solubility is detrimental to mobility. In certain embodiments, aprodrug is an ester. In certain such embodiments, the ester ismetabolically hydrolyzed to carboxylic acid upon administration. Incertain instances the carboxylic acid containing compound is thecorresponding active form. In certain embodiments, a prodrug comprises ashort peptide (polyaminoacid) bound to an acid group. In certain of suchembodiments, the peptide is cleaved upon administration to form thecorresponding active form.

In certain embodiments, the present disclosure provides compositions andmethods for reducing the amount or activity of a target nucleic acid ina cell. In certain embodiments, the cell is in an animal. In certainembodiments, the animal is a mammal. In certain embodiments, the animalis a rodent. In certain embodiments, the animal is a primate. In certainembodiments, the animal is a non-human primate. In certain embodiments,the animal is a human.

In certain embodiments, the present disclosure provides methods ofadministering a pharmaceutical composition comprising an oligonucleotideof the present disclosure to an animal. Suitable administration routesinclude, but are not limited to, oral, rectal, transmucosal, intestinal,enteral, topical, suppository, through inhalation, intrathecal,intracerebroventricular, intraperitoneal, intranasal, intraocular,intratumoral, and parenteral (e.g., intravenous, intramuscular,intramedullary, and subcutaneous). In certain embodiments,pharmaceutical intrathecals are administered to achieve local ratherthan systemic exposures. For example, pharmaceutical compositions may beinjected directly in the area of desired effect (e.g., into the liver).

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar (2′-OH for thenatural 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but notlimited to those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified RNAand/or DNA, including, but not limited to such nucleic acids havingmodified nucleobases. By way of further example and without limitation,an oligonucleotide having the nucleobase sequence “ATCGATCG” encompassesany oligonucleotides having such nucleobase sequence, whether modifiedor unmodified, including, but not limited to, such compounds comprisingRNA bases, such as those having sequence “AUCGAUCG” and those havingsome DNA bases and some RNA bases such as “AUCGATCG” andoligonucleotides having other modified bases, such as “AT^(me)CGAUCG,”wherein ^(me)C indicates a cytosine base comprising a methyl group atthe 5-position.

EXAMPLES

The following examples illustrate certain embodiments of the presentdisclosure and are not limiting. Moreover, where specific embodimentsare provided, the inventors have contemplated generic application ofthose specific embodiments. For example, disclosure of anoligonucleotide having a particular motif provides reasonable supportfor additional oligonucleotides having the same or similar motif. And,for example, where a particular high-affinity modification appears at aparticular position, other high-affinity modifications at the sameposition are considered suitable, unless otherwise indicated.

Example 1: General Method for the Preparation of Phosphoramidites,Compounds 1, 1a and 2

Bx is a heterocyclic base;

Compounds 1, 1a and 2 were prepared as per the procedures well known inthe art as described in the specification herein (see Seth et al.,Bioorg. Med. Chem., 2011, 21(4), 1122-1125, J. Org. Chem., 2010, 75(5),1569-1581, Nucleic Acids Symposium Series, 2008, 52(1), 553-554); andalso see published PCT International Applications (WO 2011/115818, WO2010/077578, WO2010/036698, WO2009/143369, WO 2009/006478, and WO2007/090071), and U.S. Pat. No. 7,569,686).

Example 2: Preparation of Compound 7

Compounds 3(2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-Dgalactopyranose orgalactosamine pentaacetate) is commercially available. Compound 5 wasprepared according to published procedures (Weber et al., J. Med. Chem.,1991, 34, 2692).

Example 3: Preparation of Compound 11

Compounds 8 and 9 are commercially available.

Example 4: Preparation of Compound 18

Compound 11 was prepared as per the procedures illustrated in Example 3.Compound 14 is commercially available. Compound 17 was prepared usingsimilar procedures reported by Rensen et al., J. Med. Chem., 2004, 47,5798-5808.

Example 5: Preparation of Compound 23

Compounds 19 and 21 are commercially available.

Example 6: Preparation of Compound 24

Compounds 18 and 23 were prepared as per the procedures illustrated inExamples 4 and 5.

Example 7: Preparation of Compound 25

Compound 24 was prepared as per the procedures illustrated in Example 6.

Example 8: Preparation of Compound 26

Compound 24 is prepared as per the procedures illustrated in Example 6.

Example 9: General Preparation of Conjugated ASOs Comprising GalNAc₃-1at the 3′ Terminus, Compound 29

Wherein the protected GalNAc₃-1 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-1(GalNAc₃-1_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-1_(a) has the formula:

The solid support bound protected GalNAc₃-1, Compound 25, was preparedas per the procedures illustrated in Example 7. Oligomeric Compound 29comprising GalNAc₃-1 at the 3′ terminus was prepared using standardprocedures in automated DNA/RNA synthesis (see Dupouy et al., Angew.Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks,Compounds 1 and 1a were prepared as per the procedures illustrated inExample 1. The phosphoramidites illustrated are meant to berepresentative and not intended to be limiting as other phosphoramiditebuilding blocks can be used to prepare oligomeric compounds having apredetermined sequence and composition. The order and quantity ofphosphoramidites added to the solid support can be adjusted to preparegapped oligomeric compounds as described herein. Such gapped oligomericcompounds can have predetermined composition and base sequence asdictated by any given target.

Example 10: General Preparation Conjugated ASOs Comprising GalNAc₃-1 atthe 5′ Terminus, Compound 34

The Unylinker™ 30 is commercially available. Oligomeric Compound 34comprising a GalNAc₃-1 cluster at the 5′ terminus is prepared usingstandard procedures in automated DNA/RNA synthesis (see Dupouy et al.,Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite buildingblocks, Compounds 1 and 1a were prepared as per the proceduresillustrated in Example 1. The phosphoramidites illustrated are meant tobe representative and not intended to be limiting as otherphosphoramidite building blocks can be used to prepare an oligomericcompound having a predetermined sequence and composition. The order andquantity of phosphoramidites added to the solid support can be adjustedto prepare gapped oligomeric compounds as described herein. Such gappedoligomeric compounds can have predetermined composition and basesequence as dictated by any given target.

Example 11: Preparation of Compound 39

Compounds 4, 13 and 23 were prepared as per the procedures illustratedin Examples 2, 4, and 5. Compound 35 is prepared using similarprocedures published in Rouchaud et al., Eur. J. Org. Chem., 2011, 12,2346-2353.

Example 12: Preparation of Compound 40

Compound 38 is prepared as per the procedures illustrated in Example 11.

Example 13: Preparation of Compound 44

Compounds 23 and 36 are prepared as per the procedures illustrated inExamples 5 and 11. Compound 41 is prepared using similar procedurespublished in WO 2009082607.

Example 14: Preparation of Compound 45

Compound 43 is prepared as per the procedures illustrated in Example 13.

Example 15: Preparation of Compound 47

Compound 46 is commercially available.

Example 16: Preparation of Compound 53

Compounds 48 and 49 are commercially available. Compounds 17 and 47 areprepared as per the procedures illustrated in Examples 4 and 15.

Example 17: Preparation of Compound 54

Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 18: Preparation of Compound 55

Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 19: General Method for the Preparation of Conjugated ASOsComprising GalNAc₃-1 at the 3′ Position Via Solid Phase Techniques(Preparation of ISIS 647535, 647536 and 651900)

Unless otherwise stated, all reagents and solutions used for thesynthesis of oligomeric compounds are purchased from commercial sources.Standard phosphoramidite building blocks and solid support are used forincorporation nucleoside residues which include for example T, A, G, and^(m)C residues. A 0.1 M solution of phosphoramidite in anhydrousacetonitrile was used for β-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale)or on GE Healthcare Bioscience ÄKTA oligopilot synthesizer (40-200 μmolscale) by the phosphoramidite coupling method on an GalNAc₃-1 loadedVIMAD solid support (110 μmol/g, Guzaev et al., 2003) packed in thecolumn. For the coupling step, the phosphoramidites were delivered 4fold excess over the loading on the solid support and phosphoramiditecondensation was carried out for 10 min. All other steps followedstandard protocols supplied by the manufacturer. A solution of 6%dichloroacetic acid in toluene was used for removing dimethoxytrityl(DMT) group from 5′-hydroxyl group of the nucleotide.4,5-Dicyanoimidazole (0.7 M) in anhydrous CH₃CN was used as activatorduring coupling step. Phosphorothioate linkages were introduced bysulfurization with 0.1 M solution of xanthane hydride in 1:1pyridine/CH₃CN for a contact time of 3 minutes. A solution of 20%tert-butylhydroperoxide in CH₃CN containing 6% water was used as anoxidizing agent to provide phosphodiester internucleoside linkages witha contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphateprotecting groups were deprotected using a 1:1 (v/v) mixture oftriethylamine and acetonitrile with a contact time of 45 minutes. Thesolid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %)and heated at 55° C. for 6 h.

The unbound ASOs were then filtered and the ammonia was boiled off. Theresidue was purified by high pressure liquid chromatography on a stronganion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm,2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 M NaBrin A, 0-40% of B in 60 min, flow 14 mL min-1, =260 nm). The residue wasdesalted by HPLC on a reverse phase column to yield the desired ASOs inan isolated yield of 15-30% based on the initial loading on the solidsupport. The ASOs were characterized by ion-pair-HPLC coupled MSanalysis with Agilent 1100 MSD system.

Antisense oligonucleotides not comprising a conjugate were synthesizedusing standard oligonucleotide synthesis procedures well known in theart.

Using these methods, three separate antisense compounds targeting ApoCIII were prepared. As summarized in Table 17, below, each of the threeantisense compounds targeting ApoC III had the same nucleobase sequence;ISIS 304801 is a 5-10-5 MOE gapmer having all phosphorothioate linkages;ISIS 647535 is the same as ISIS 304801, except that it had a GalNAc₃-1conjugated at its 3′ end; and ISIS 647536 is the same as ISIS 647535except that certain internucleoside linkages of that compound arephosphodiester linkages. As further summarized in Table 17, two separateantisense compounds targeting SRB-1 were synthesized. ISIS 440762 was a2-10-2 cEt gapmer with all phosphorothioate internucleoside linkages;ISIS 651900 is the same as ISIS 440762, except that it included aGalNAc₃-1 at its 3′-end.

TABLE 17 Modified ASO targeting ApoC III and SRB-1 SEQ CalCd Observed IDASO Sequence (5′ to 3′) Target Mass Mass No. ISIS A_(es)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ApoC7165.4 7164.4 135 304801 III ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′) - ApoC 9239.5 9237.8136 647535 GalNAc ₃ -1 _(a) III ISIS A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(eo) A _(do′) - ApoC 9142.9 9140.8136 647536 GalNAc ₃ -1 _(a) III ISIS T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)SRB-1 4647.0 4646.4 137 440762 ISIS T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A_(do′) -GalNAc ₃ -1 _(a) SRB-1 6721.1 6719.4 138 651900Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside(e.g. cEt); “s” indicates phosphorothioate internucleoside linkages(PS); “o” indicates phosphodiester internucleoside linkages (PO); and“o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates5-methylcytosines. “GalNAc₃-1” indicates a conjugate group having thestructure shown previously in Example 9. Note that GalNAc₃-1 comprises acleavable adenosine which links the ASO to remainder of the conjugate,which is designated “GalNAc₃-1_(a).” This nomenclature is used in theabove table to show the full nucleobase sequence, including theadenosine, which is part of the conjugate. Thus, in the above table, thesequences could also be listed as ending with “GalNAc₃-1” with the“A_(do)” omitted. This convention of using the subscript “a” to indicatethe portion of a conjugate group lacking a cleavable nucleoside orcleavable moiety is used throughout these Examples. This portion of aconjugate group lacking the cleavable moiety is referred to herein as a“cluster” or “conjugate cluster” or “GalNAc₃ cluster.” In certaininstances it is convenient to describe a conjugate group by separatelyproviding its cluster and its cleavable moiety.

Example 20: Dose-Dependent Antisense Inhibition of Human ApoC III inhuApoC III Transgenic Mice

ISIS 304801 and ISIS 647535, each targeting human ApoC III and describedabove, were separately tested and evaluated in a dose-dependent studyfor their ability to inhibit human ApoC III in human ApoC III transgenicmice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/darkcycle and fed ad libitum Teklad lab chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. ASOs were prepared in PBS and sterilized by filteringthrough a 0.2 micron filter. ASOs were dissolved in 0.9% PBS forinjection.

Human ApoC III transgenic mice were injected intraperitoneally once aweek for two weeks with ISIS 304801 or 647535 at 0.08, 0.25, 0.75, 2.25or 6.75 μmol/kg or with PBS as a control. Each treatment group consistedof 4 animals. Forty-eight hours after the administration of the lastdose, blood was drawn from each mouse and the mice were sacrificed andtissues were collected.

ApoC III mRNA Analysis

ApoC III mRNA levels in the mice's livers were determined usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. ApoC IIImRNA levels were determined relative to total RNA (using Ribogreen),prior to normalization to PBS-treated control. The results below arepresented as the average percent of ApoC III mRNA levels for eachtreatment group, normalized to PBS-treated control and are denoted as “%PBS”. The half maximal effective dosage (ED₅₀) of each ASO is alsopresented in Table 18, below.

As illustrated, both antisense compounds reduced ApoC III RNA relativeto the PBS control. Further, the antisense compound conjugated toGalNAc₃-1 (ISIS 647535) was substantially more potent than the antisensecompound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 18 Effect of ASO treatment on ApoC III mRNA levels in human ApoCIII transgenic mice Inter- nucleoside SEQ Dose % ED₅₀ 3’ linkage/ ID ASO(μmol/kg) PBS (μmol/kg) Conjugate Length No. PBS 0 100 — — — ISIS 0.0895 0.77 None PS/20 135 304801 0.75 42 2.25 32 6.75 19 ISIS 0.08 50 0.074GalNAc₃-1 PS/20 136 647535 0.75 15 2.25 17 6.75 8ApoC III Protein Analysis (Turbidometric Assay)

Plasma ApoC III protein analysis was determined using proceduresreported by Graham et al, Circulation Research, published online beforeprint Mar. 29, 2013.

Approximately 100 μl of plasma isolated from mice was analyzed withoutdilution using an Olympus Clinical Analyzer and a commercially availableturbidometric ApoC III assay (Kamiya, Cat # KAI-006, Kamiya Biomedical,Seattle, Wash.). The assay protocol was performed as described by thevendor.

As shown in the Table 19 below, both antisense compounds reduced ApoCIII protein relative to the PBS control. Further, the antisense compoundconjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent thanthe antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 19 Effect of ASO treatment on ApoC III plasma protein levels inhuman ApoC III transgenic mice Inter- nucleoside SEQ Dose % ED₅₀ 3’Linkage/ ID ASO (μmol/kg) PBS (μmol/kg) Conjugate Length No. PBS 0 100 —— — ISIS 0.08 86 0.73 None PS/20 135 304801 0.75 51 2.25 23 6.75 13 ISIS0.08 72 0.19 GalNAc₃-1 PS/20 136 647535 0.75 14 2.25 12 6.75 11

Plasma triglycerides and cholesterol were extracted by the method ofBligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J. Biochem. Physiol.37: 911-917, 1959) (Bligh, E and Dyer, W, Can J Biochem Physiol, 37,911-917, 1959) (Bligh, E and Dyer, W, Can J Biochem Physiol, 37,911-917, 1959) and measured by using a Beckmann Coulter clinicalanalyzer and commercially available reagents.

The triglyceride levels were measured relative to PBS injected mice andare denoted as “% PBS”. Results are presented in Table 20. Asillustrated, both antisense compounds lowered triglyceride levels.Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535)was substantially more potent than the antisense compound lacking theGalNAc₃-1 conjugate (ISIS 304801).

TABLE 20 Effect of ASO treatment on triglyceride levels in transgenicmice Inter- nucleoside SEQ Dose % ED₅₀ 3’ Linkage/ ID ASO (μmol/kg) PBS(μmol/kg) Conjugate Length No. PBS 0 100 — — — ISIS 0.08 87 0.63 NonePS/20 135 304801 0.75 46 2.25 21 6.75 12 ISIS 0.08 65 0.13 GalNAc₃-1PS/20 136 647535 0.75 9 2.25 8 6.75 9

Plasma samples were analyzed by HPLC to determine the amount of totalcholesterol and of different fractions of cholesterol (HDL and LDL).Results are presented in Tables 21 and 22. As illustrated, bothantisense compounds lowered total cholesterol levels; both lowered LDL;and both raised HDL. Further, the antisense compound conjugated toGalNAc₃-1 (ISIS 647535) was substantially more potent than the antisensecompound lacking the GalNAc₃-1 conjugate (ISIS 304801). An increase inHDL and a decrease in LDL levels is a cardiovascular beneficial effectof antisense inhibition of ApoC III.

TABLE 21 Effect of ASO treatment on total cholesterol levels intransgenic mice Total Dose Cholesterol 3′ Internucleoside SEQ ASO(μmol/kg) (mg/dL) Conjugate Linkage/Length ID No. PBS 0 257 — — ISIS0.08 226 None PS/20 135 304801 0.75 164 2.25 110 6.75 82 ISIS 0.08 230GalNAc₃-1 PS/20 136 647535 0.75 82 2.25 86 6.75 99

TABLE 22 Effect of ASO treatment on HDL and LDL cholesterol levels intransgenic mice Inter- nucleoside SEQ Dose HDL LDL 3’ Linkage/ ID ASO(μmol/kg) (mg/dL) (mg/dL) Conjugate Length No. PBS 0 17 28 — — ISIS 0.0817 23 None PS/20 135 304801 0.75 27 12 2.25 50 4 6.75 45 2 ISIS 0.08 2121 GalNAc₃-1 PS/20 136 647535 0.75 44 2 2.25 50 2 6.75 58 2Pharmacokinetics Analysis (PK)

The PK of the ASOs was also evaluated. Liver and kidney samples wereminced and extracted using standard protocols. Samples were analyzed onMSD1 utilizing IP-HPLC-MS. The tissue level (μg/g) of full-length ISIS304801 and 647535 was measured and the results are provided in Table 23.As illustrated, liver concentrations of total full-length antisensecompounds were similar for the two antisense compounds. Thus, eventhough the GalNAc₃-1-conjugated antisense compound is more active in theliver (as demonstrated by the RNA and protein data above), it is notpresent at substantially higher concentration in the liver. Indeed, thecalculated EC₅₀ (provided in Table 23) confirms that the observedincrease in potency of the conjugated compound cannot be entirelyattributed to increased accumulation. This result suggests that theconjugate improved potency by a mechanism other than liver accumulationalone, possibly by improving the productive uptake of the antisensecompound into cells.

The results also show that the concentration of GalNAc₃-1 conjugatedantisense compound in the kidney is lower than that of antisensecompound lacking the GalNAc conjugate. This has several beneficialtherapeutic implications. For therapeutic indications where activity inthe kidney is not sought, exposure to kidney risks kidney toxicitywithout corresponding benefit. Moreover, high concentration in kidneytypically results in loss of compound to the urine resulting in fasterclearance. Accordingly, for non-kidney targets, kidney accumulation isundesired. These data suggest that GalNAc₃-1 conjugation reduces kidneyaccumulation.

TABLE 23 PK analysis of ASO treatment in transgenic mice Inter- Livernucleoside SEQ Dose Liver Kidney EC₅₀ 3’ (Linkage/ ID ASO (μmol/kg)(μg/g) (μg/g) (μg/g) Conjugate Length) No. ISIS 0.1 5.2 2.1 53 NonePS/20 135 304801 0.8 62.8 119.6 2.3 142.3 191.5 6.8 202.3 337.7 ISIS 0.13.8 0.7 3.8 GalNAc₃-1 PS/20 136 647535 0.8 72.7 34.3 2.3 106.8 111.4 6.8237.2 179.3

Metabolites of ISIS 647535 were also identified and their masses wereconfirmed by high resolution mass spectrometry analysis. The cleavagesites and structures of the observed metabolites are shown below. Therelative % of full length ASO was calculated using standard proceduresand the results are presented in Table 23a. The major metabolite of ISIS647535 was full-length ASO lacking the entire conjugate (i.e. ISIS304801), which results from cleavage at cleavage site A, shown below.Further, additional metabolites resulting from other cleavage sites werealso observed. These results suggest that introducing other cleabablebonds such as esters, peptides, disulfides, phosphoramidates oracyl-hydrazones between the GalNAc₃-1 sugar and the ASO, which can becleaved by enzymes inside the cell, or which may cleave in the reductiveenvironment of the cytosol, or which are labile to the acidic pH insideendosomes and lyzosomes, can also be useful.

TABLE 23a Observed full length metabolites of ISIS 647535 Metabolite ASOCleavage site Relative % 1 ISIS 304801 A 36.1 2 ISIS 304801 + dA B 10.53 ISIS 647535 minus [3 GalNAc] C 16.1 4 ISIS 647535 minus D 17.6 [3GalNAc + 1 5-hydroxy-pentanoic acid tether] 5 ISIS 647535 minus D 9.9 [2GalNAc + 2 5-hydroxy-pentanoic acid tether] 6 ISIS 647535 minus D 9.8 [3GalNAc + 3 5-hydroxy-pentanoic acid tether]

Example 21: Antisense Inhibition of Human ApoC III in Human ApoC IIITransgenic Mice in Single Administration Study

ISIS 304801, 647535 and 647536 each targeting human ApoC III anddescribed in Table 17, were further evaluated in a single administrationstudy for their ability to inhibit human ApoC III in human ApoC IIItransgenic mice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/darkcycle and fed ad libitum Teklad lab chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. ASOs were prepared in PBS and sterilized by filteringthrough a 0.2 micron filter. ASOs were dissolved in 0.9% PBS forinjection.

Human ApoC III transgenic mice were injected intraperitoneally once atthe dosage shown below with ISIS 304801, 647535 or 647536 (describedabove) or with PBS treated control. The treatment group consisted of 3animals and the control group consisted of 4 animals. Prior to thetreatment as well as after the last dose, blood was drawn from eachmouse and plasma samples were analyzed. The mice were sacrificed 72hours following the last administration.

Samples were collected and analyzed to determine the ApoC III mRNA andprotein levels in the liver; plasma triglycerides; and cholesterol,including HDL and LDL fractions were assessed as described above(Example 20). Data from those analyses are presented in Tables 24-28,below. Liver transaminase levels, alanine aminotransferase (ALT) andaspartate aminotransferase (AST), in serum were measured relative tosaline injected mice using standard protocols. The ALT and AST levelsshowed that the antisense compounds were well tolerated at alladministered doses.

These results show improvement in potency for antisense compoundscomprising a GalNAc₃-1 conjugate at the 3′ terminus (ISIS 647535 and647536) compared to the antisense compound lacking a GalNAc₃-1 conjugate(ISIS 304801). Further, ISIS 647536, which comprises a GalNAc₃-1conjugate and some phosphodiester linkages was as potent as ISIS 647535,which comprises the same conjugate and all internucleoside linkageswithin the ASO are phosphorothioate.

TABLE 24 Effect of ASO treatment on ApoC III mRNA levels in human ApoCIII transgenic mice Inter- nucleoside SEQ Dose % ED₅₀ 3’ linkage/ ID ASO(mg/kg) PBS (mg/kg) Conjugate Length No. PBS 0 99 — — — ISIS 1 104 13.2None PS/20 135 304801 3 92 10 71 30 40 ISIS 0.3 98 1.9 GalNAc₃-1 PS/20136 647535 1 70 3 33 10 20 ISIS 0.3 103 1.7 GalNAc₃-1 PS/PO/20 136647536 1 60 3 31 10 21

TABLE 25 Effect of ASO treatment on ApoC III plasma protein levels inhuman ApoC III transgenic mice Inter- nucleoside SEQ Dose % ED₅₀ 3’Linkage/ ID ASO (mg/kg) PBS (mg/kg) Conjugate Length No. PBS 0 99 — — —ISIS 1 104 23.2 None PS/20 135 304801 3 92 10 71 30 40 ISIS 0.3 98 2.1GalNAc₃-1 PS/20 136 647535 1 70 3 33 10 20 ISIS 0.3 103 1.8 GalNAc₃-1PS/PO/20 136 647536 1 60 3 31 10 21

TABLE 26 Effect of ASO treatment on triglyceride levels in transgenicmice Inter- nucleoside SEQ Dose % ED₅₀ 3’ Linkage/ ID ASO (mg/kg) PBS(mg/kg) Conjugate Length No. PBS 0 98 — — — ISIS 1 80 29.1 None PS/20135 304801 3 92 10 70 30 47 ISIS 0.3 100 2.2 GalNAc₃-1 PS/20 136 6475351 70 3 34 10 23 ISIS 0.3 95 19 GalNAc₃-1 PS/PO/20 136 647536 1 66 3 3110 23

TABLE 27 Effect of ASO treatment on total cholesterol levels intransgenic mice Dose 3′ Internucleoside SEQ ID ASO (mg/kg) % PBSConjugate Linkage/Length No. PBS 0 96 — — ISIS 1 104 None PS/20 135304801 3 96 10 86 30 72 ISIS 0.3 93 GalNAc₃-1 PS/20 136 647535 1 85 3 6110 53 ISIS 0.3 115 GalNAc₃-1 PS/PO/20 136 647536 1 79 3 51 10 54

TABLE 28 Effect of ASO treatment on HDL and LDL cholesterol levels intransgenic mice Inter- nucleoside SEQ Dose HDL LDL 3’ Linkage/ ID ASO(mg/kg) % PBS % PBS Conjugate Length No. PBS 0 131 90 — — ISIS 1 130 72None PS/20 135 304801 3 186 79 10 226 63 30 240 46 ISIS 0.3 98 86GalNAc₃-1 PS/20 136 647535 1 214 67 3 212 39 10 218 35 ISIS 0.3 143 89GalNAc₃-1 PS/PO/20 136 647536 1 187 56 3 213 33 10 221 34

These results confirm that the GalNAc₃-1 conjugate improves potency ofan antisense compound. The results also show equal potency of aGalNAc₃-1 conjugated antisense compounds where the antisenseoligonucleotides have mixed linkages (ISIS 647536 which has sixphosphodiester linkages) and a full phosphorothioate version of the sameantisense compound (ISIS 647535).

Phosphorothioate linkages provide several properties to antisensecompounds. For example, they resist nuclease digestion and they bindproteins resulting in accumulation of compound in the liver, rather thanin the kidney/urine. These are desirable properties, particularly whentreating an indication in the liver. However, phosphorothioate linkageshave also been associated with an inflammatory response. Accordingly,reducing the number of phosphorothioate linkages in a compound isexpected to reduce the risk of inflammation, but also lowerconcentration of the compound in liver, increase concentration in thekidney and urine, decrease stability in the presence of nucleases, andlower overall potency. The present results show that a GalNAc₃-1conjugated antisense compound where certain phosphorothioate linkageshave been replaced with phosphodiester linkages is as potent against atarget in the liver as a counterpart having full phosphorothioatelinkages. Such compounds are expected to be less proinflammatory (SeeExample 24 describing an experiment showing reduction of PS results inreduced inflammatory effect).

Example 22: Effect of GalNAc₃-1 Conjugated Modified ASO Targeting SRB-1In Vivo

ISIS 440762 and 651900, each targeting SRB-1 and described in Table 17,were evaluated in a dose-dependent study for their ability to inhibitSRB-1 in Balb/c mice.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 440762,651900 or with PBS treated control. Each treatment group consisted of 4animals. The mice were sacrificed 48 hours following the finaladministration to determine the SRB-1 mRNA levels in liver usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNAlevels were determined relative to total RNA (using Ribogreen), prior tonormalization to PBS-treated control. The results below are presented asthe average percent of SRB-1 mRNA levels for each treatment group,normalized to PBS-treated control and is denoted as “% PBS”.

As illustrated in Table 29, both antisense compounds lowered SRB-1 mRNAlevels. Further, the antisense compound comprising the GalNAc₃-1conjugate (ISIS 651900) was substantially more potent than the antisensecompound lacking the GalNAc₃-1 conjugate (ISIS 440762). These resultsdemonstrate that the potency benefit of GalNAc₃-1 conjugates areobserved using antisense oligonucleotides complementary to a differenttarget and having different chemically modified nucleosides, in thisinstance modified nucleosides comprise constrained ethyl sugar moieties(a bicyclic sugar moiety).

TABLE 29 Effect of ASO treatment on SRB-1 mRNA levels in Balb/c miceInter- nucleoside SEQ Dose Liver ED₅₀ 3’ linkage/ ID ASO (mg/kg) % PBS(mg/kg) Conjugate Length No. PBS 0 100 — — ISIS 0.7 85 2.2 None PS/14137 440762 2 55 7 12 20 3 ISIS 0.07 98 0.3 GalNAc₃-1 PS/14 138 6519000.2 63 0.7 20 2 6 7 5

Example 23: Human Peripheral Blood Mononuclear Cells (hPBMC) AssayProtocol

The hPBMC assay was performed using BD Vautainer CPT tube method. Asample of whole blood from volunteered donors with informed consent atUS HealthWorks clinic (Faraday & El Camino Real, Carlsbad) was obtainedand collected in 4-15 BD Vacutainer CPT 8 ml tubes (VWR Cat. #BD362753). The approximate starting total whole blood volume in the CPTtubes for each donor was recorded using the PBMC assay data sheet.

The blood sample was remixed immediately prior to centrifugation bygently inverting tubes 8-10 times. CPT tubes were centrifuged at rt(18-25° C.) in a horizontal (swing-out) rotor for 30 min. at 1500-1800RCF with brake off (2700 RPM Beckman Allegra 6R). The cells wereretrieved from the buffy coat interface (between Ficoll and polymer gellayers); transferred to a sterile 50 ml conical tube and pooled up to 5CPT tubes/50 ml conical tube/donor. The cells were then washed twicewith PBS (Ca⁺⁺, Mg⁺⁺ free; GIBCO). The tubes were topped up to 50 ml andmixed by inverting several times. The sample was then centrifuged at330×g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) andaspirated as much supernatant as possible without disturbing pellet. Thecell pellet was dislodged by gently swirling tube and resuspended cellsin RPMI+10% FBS+pen/strep (˜1 ml/10 ml starting whole blood volume). A60 μl sample was pipette into a sample vial (Beckman Coulter) with 600μl VersaLyse reagent (Beckman Coulter Cat # A09777) and was gentlyvortexed for 10-15 sec. The sample was allowed to incubate for 10 min.at rt and being mixed again before counting. The cell suspension wascounted on Vicell XR cell viability analyzer (Beckman Coulter) usingPBMC cell type (dilution factor of 1:11 was stored with otherparameters). The live cell/ml and viability were recorded. The cellsuspension was diluted to 1×10⁷ live PBMC/ml in RPMI+10% FBS+pen/strep.

The cells were plated at 5×10⁵ in 50 μl/well of 96-well tissue cultureplate (Falcon Microtest). 50 μl/well of 2× concentration oligos/controlsdiluted in RPMI+10% FBS+pen/strep. was added according to experimenttemplate (100 μl/well total). Plates were placed on the shaker andallowed to mix for approx. 1 min. After being incubated for 24 hrs at37° C.; 5% CO₂, the plates were centrifuged at 400×g for 10 minutesbefore removing the supernatant for MSD cytokine assay (i.e. human IL-6,IL-10, IL-8 and MCP-1).

Example 24: Evaluation of Proinflammatory Effects in hPBMC Assay forGalNAc₃-1 Conjugated ASOs

The antisense oligonucleotides (ASOs) listed in Table 30 were evaluatedfor proinflammatory effect in hPBMC assay using the protocol describedin Example 23. ISIS 353512 is an internal standard known to be a highresponder for IL-6 release in the assay. The hPBMCs were isolated fromfresh, volunteered donors and were treated with ASOs at 0, 0.0128,0.064, 0.32, 1.6, 8, 40 and 200 μM concentrations. After a 24 hrtreatment, the cytokine levels were measured.

The levels of IL-6 were used as the primary readout. The EC₅₀ andE_(max) was calculated using standard procedures. Results are expressedas the average ratio of E_(max)/EC₅₀ from two donors and is denoted as“E_(max)/EC₅₀.” The lower ratio indicates a relative decrease in theproinflammatory response and the higher ratio indicates a relativeincrease in the proinflammatory response.

With regard to the test compounds, the least proinflammatory compoundwas the PS/PO linked ASO (ISIS 616468). The GalNAc₃-1 conjugated ASO,ISIS 647535 was slightly less proinflammatory than its non-conjugatedcounterpart ISIS 304801. These results indicate that incorporation ofsome PO linkages reduces proinflammatory reaction and addition of aGalNAc₃-1 conjugate does not make a compound more proinflammatory andmay reduce proinflammatory response. Accordingly, one would expect thatan antisense compound comprising both mixed PS/PO linkages and aGalNAc₃-1 conjugate would produce lower proinflammatory responsesrelative to full PS linked antisense compound with or without aGalNAc₃-1 conjugate. These results show that GalNAc₃-1 conjugatedantisense compounds, particularly those having reduced PS content areless proinflammatory.

Together, these results suggest that a GalNAc₃-1 conjugated compound,particularly one with reduced PS content, can be administered at ahigher dose than a counterpart full PS antisense compound lacking aGalNAc₃-1 conjugate. Since half-life is not expected to be substantiallydifferent for these compounds, such higher administration would resultin less frequent dosing. Indeed such administration could be even lessfrequent, because the GalNAc₃-1 conjugated compounds are more potent(See Examples 20-22) and re-dosing is necessary once the concentrationof a compound has dropped below a desired level, where such desiredlevel is based on potency.

TABLE 30 Modified ASOs SEQ ID ASO Sequence (5′ to 3′) Target No. ISISG_(es) ^(m)C_(es)T_(es)G_(es)A_(es)T_(ds)T_(ds)A_(ds)G_(ds)A_(ds)G_(ds)TNFα 139 104838 A_(ds)G_(ds)A_(ds)G_(ds)G_(es)T_(es) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) ISIS T_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(ds)A_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) CRP 140 353512G_(ds)A_(ds)G_(ds)A_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(es)G_(es)G_(e) ISISA_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)ApoC III 135 304801 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC III 136 647535 ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′)-GalNAc ₃ -1 _(a) ISIS A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC III 135 616468 ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(e)

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside(e.g. cEt); “s” indicates phosphorothioate internucleoside linkages(PS); “o” indicates phosphodiester internucleoside linkages (PO); and“o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates5-methylcytosines. “A_(do′)-GalNAc₃-1_(a)” indicates a conjugate havingthe structure GalNAc₃-1 shown in Example 9 attached to the 3′-end of theantisense oligonucleotide, as indicated.

TABLE 31 Proinflammatory Effect of ASOs targeting ApoC III in hPBMCassay EC₅₀ E_(max) 3′ Internucleoside SEQ ID ASO (μm) (μm) E_(max)/EC₅₀Conjugate Linkage/Length No. ISIS 353512 0.01 265.9 26,590 None PS/20140 (high responder) ISIS 304801 0.07 106.55 1,522 None PS/20 135 ISIS647535 0.12 138 1,150 GalNAc ₃ -1 PS/20 136 ISIS 616468 0.32 71.52 224None PS/PO/20 135

Example 25: Effect of GalNAc₃-1 Conjugated Modified ASO Targeting HumanApoC III In Vitro

ISIS 304801 and 647535 described above were tested in vitro. Primaryhepatocyte cells from transgenic mice at a density of 25,000 cells perwell were treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 and 20 μMconcentrations of modified oligonucleotides. After a treatment period ofapproximately 16 hours, RNA was isolated from the cells and mRNA levelswere measured by quantitative real-time PCR and the hApoC III mRNAlevels were adjusted according to total RNA content, as measured byRIBOGREEN.

The IC₅₀ was calculated using the standard methods and the results arepresented in Table 32. As illustrated, comparable potency was observedin cells treated with ISIS 647535 as compared to the control, ISIS304801.

TABLE 32 Modified ASO targeting human ApoC III in primary hepatocytes 3′Internucleoside SEQ ASO IC₅₀ (μM) Conjugate linkage/Length ID No. ISIS0.44 None PS/20 135 304801 ISIS 0.31 GalNAc₃-1 PS/20 136 647535

In this experiment, the large potency benefits of GalNAc₃-1 conjugationthat are observed in vivo were not observed in vitro. Subsequent freeuptake experiments in primary hepatocytes in vitro did show increasedpotency of oligonucleotides comprising various GalNAc conjugatesrelative to oligonucleotides that lacking the GalNAc conjugate. (seeExamples 60, 82, and 92)

Example 26: Effect of PO/PS Linkages on ApoC III ASO Activity

Human ApoC III transgenic mice were injected intraperitoneally once at25 mg/kg of ISIS 304801, or ISIS 616468 (both described above) or withPBS treated control once per week for two weeks. The treatment groupconsisted of 3 animals and the control group consisted of 4 animals.Prior to the treatment as well as after the last dose, blood was drawnfrom each mouse and plasma samples were analyzed. The mice weresacrificed 72 hours following the last administration.

Samples were collected and analyzed to determine the ApoC III proteinlevels in the liver as described above (Example 20). Data from thoseanalyses are presented in Table 33, below.

These results show reduction in potency for antisense compounds withPO/PS (ISIS 616468) in the wings relative to full PS (ISIS 304801).

TABLE 33 Effect of ASO treatment on ApoC III protein levels in humanApoC III transgenic mice Dose 3′ Internucleoside SEQ ID ASO (mg/kg) %PBS Conjugate linkage/Length No. PBS  0 99 — — ISIS 25 24 None Full PS135 304801 mg/kg/wk for 2 wks ISIS 25 40 None 14 PS/6 PO 135 616468mg/kg/wk for 2 wks

Example 27: Compound 56

Compound 56 is commercially available from Glen Research or may beprepared according to published procedures reported by Shchepinov etal., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 28: Preparation of Compound 60

Compound 4 was prepared as per the procedures illustrated in Example 2.Compound 57 is commercially available. Compound 60 was confirmed bystructural analysis.

Compound 57 is meant to be representative and not intended to belimiting as other monoprotected substituted or unsubstituted alkyl diolsincluding but not limited to those presented in the specification hereincan be used to prepare phosphoramidites having a predeterminedcomposition.

Example 29: Preparation of Compound 63

Compounds 61 and 62 are prepared using procedures similar to thosereported by Tober et al., Eur. J. Org. Chem., 2013, 3, 566-577; andJiang et al., Tetrahedron, 2007, 63(19), 3982-3988.

Alternatively, Compound 63 is prepared using procedures similar to thosereported in scientific and patent literature by Kim et al., Synlett,2003, 12, 1838-1840; and Kim et al., published PCT InternationalApplication, WO 2004063208.

Example 30: Preparation of Compound 63b

Compound 63a is prepared using procedures similar to those reported byHanessian et al., Canadian Journal of Chemistry, 1996, 74(9), 1731-1737.

Example 31: Preparation of Compound 63d

Compound 63c is prepared using procedures similar to those reported byChen et al., Chinese Chemical Letters, 1998, 9(5), 451-453.

Example 32: Preparation of Compound 67

Compound 64 was prepared as per the procedures illustrated in Example 2.Compound 65 is prepared using procedures similar to those reported by Oret al., published PCT International Application, WO 2009003009. Theprotecting groups used for Compound 65 are meant to be representativeand not intended to be limiting as other protecting groups including butnot limited to those presented in the specification herein can be used.

Example 33: Preparation of Compound 70

Compound 64 was prepared as per the procedures illustrated in Example 2.Compound 68 is commercially available. The protecting group used forCompound 68 is meant to be representative and not intended to belimiting as other protecting groups including but not limited to thosepresented in the specification herein can be used.

Example 34: Preparation of Compound 75a

Compound 75 is prepared according to published procedures reported byShchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 35: Preparation of Compound 79

Compound 76 was prepared according to published procedures reported byShchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 36: Preparation of Compound 79a

Compound 77 is prepared as per the procedures illustrated in Example 35.

Example 37: General Method for the Preparation of Conjugated OligomericCompound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′Terminus Via Solid Support (Method I)

wherein GalNAc₃-2 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-2(GalNAc₃-2_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-2_(a) has the formula:

The VIMAD-bound oligomeric compound 79b was prepared using standardprocedures for automated DNA/RNA synthesis (see Dupouy et al., Angew.Chem. Int. Ed., 2006, 45, 3623-3627). The phosphoramidite Compounds 56and 60 were prepared as per the procedures illustrated in Examples 27and 28, respectively. The phosphoramidites illustrated are meant to berepresentative and not intended to be limiting as other phosphoramiditebuilding blocks including but not limited those presented in thespecification herein can be used to prepare an oligomeric compoundhaving a phosphodiester linked conjugate group at the 5′ terminus. Theorder and quantity of phosphoramidites added to the solid support can beadjusted to prepare the oligomeric compounds as described herein havingany predetermined sequence and composition.

Example 38: Alternative Method for the Preparation of OligomericCompound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′Terminus (Method II)

The VIMAD-bound oligomeric compound 79b was prepared using standardprocedures for automated DNA/RNA synthesis (see Dupouy et al., Angew.Chem. Int. Ed., 2006, 45, 3623-3627). The GalNAc₃-2 clusterphosphoramidite, Compound 79 was prepared as per the proceduresillustrated in Example 35. This alternative method allows a one-stepinstallation of the phosphodiester linked GalNAc₃-2 conjugate to theoligomeric compound at the final step of the synthesis. Thephosphoramidites illustrated are meant to be representative and notintended to be limiting, as other phosphoramidite building blocksincluding but not limited to those presented in the specification hereincan be used to prepare oligomeric compounds having a phosphodiesterconjugate at the 5′ terminus. The order and quantity of phosphoramiditesadded to the solid support can be adjusted to prepare the oligomericcompounds as described herein having any predetermined sequence andcomposition.

Example 39: General Method for the Preparation of Oligomeric Compound83h Comprising a GalNAc₃-3 Conjugate at the 5′ Terminus (GalNAc₃-1Modified for 5′ End Attachment) Via Solid Support

Compound 18 was prepared as per the procedures illustrated in Example 4.Compounds 83a and 83b are commercially available. Oligomeric Compound83e comprising a phosphodiester linked hexylamine was prepared usingstandard oligonucleotide synthesis procedures. Treatment of theprotected oligomeric compound with aqueous ammonia provided the5′-GalNAc₃-3 conjugated oligomeric compound (83h).

Wherein GalNAc₃-3 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-3(GalNAc₃-3_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-3_(a) has the formula:

Example 40: General Method for the Preparation of Oligomeric Compound 89Comprising a Phosphodiester Linked GalNAc₃-4 Conjugate at the 3′Terminus Via Solid Support

Wherein GalNAc₃-4 has the structure:

Wherein CM is a cleavable moiety. In certain embodiments, cleavablemoiety is:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-4(GalNAc₃-4_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-4_(a) has the formula:

The protected Unylinker functionalized solid support Compound 30 iscommercially available. Compound 84 is prepared using procedures similarto those reported in the literature (see Shchepinov et al., NucleicAcids Research, 1997, 25(22), 4447-4454; Shchepinov et al., NucleicAcids Research, 1999, 27, 3035-3041; and Hornet et al., Nucleic AcidsResearch, 1997, 25, 4842-4849).

The phosphoramidite building blocks, Compounds 60 and 79a are preparedas per the procedures illustrated in Examples 28 and 36. Thephosphoramidites illustrated are meant to be representative and notintended to be limiting as other phosphoramidite building blocks can beused to prepare an oligomeric compound having a phosphodiester linkedconjugate at the 3′ terminus with a predetermined sequence andcomposition. The order and quantity of phosphoramidites added to thesolid support can be adjusted to prepare the oligomeric compounds asdescribed herein having any predetermined sequence and composition.

Example 41: General Method for the Preparation of ASOs Comprising aPhosphodiester Linked GalNAc₃-2 (See Example 37, Bx is Adenine)Conjugate at the 5′ Position Via Solid Phase Techniques (Preparation ofISIS 661134)

Unless otherwise stated, all reagents and solutions used for thesynthesis of oligomeric compounds are purchased from commercial sources.Standard phosphoramidite building blocks and solid support are used forincorporation nucleoside residues which include for example T, A, G, and^(m)C residues. Phosphoramidite compounds 56 and 60 were used tosynthesize the phosphodiester linked GalNAc₃-2 conjugate at the 5′terminus. A 0.1 M solution of phosphoramidite in anhydrous acetonitrilewas used for β-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale)or on GE Healthcare Bioscience ÄKTA oligopilot synthesizer (40-200 μmolscale) by the phosphoramidite coupling method on VIMAD solid support(110 μmol/g, Guzaev et al., 2003) packed in the column. For the couplingstep, the phosphoramidites were delivered at a 4 fold excess over theinitial loading of the solid support and phosphoramidite coupling wascarried out for 10 min. All other steps followed standard protocolssupplied by the manufacturer. A solution of 6% dichloroacetic acid intoluene was used for removing the dimethoxytrityl (DMT) groups from5′-hydroxyl groups of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) inanhydrous CH₃CN was used as activator during the coupling step.Phosphorothioate linkages were introduced by sulfurization with 0.1 Msolution of xanthane hydride in 1:1 pyridine/CH₃CN for a contact time of3 minutes. A solution of 20% tert-butylhydroperoxide in CH₃CN containing6% water was used as an oxidizing agent to provide phosphodiesterinternucleoside linkages with a contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphateprotecting groups were deprotected using a 20% diethylamine in toluene(v/v) with a contact time of 45 minutes. The solid-support bound ASOswere suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. for6 h. The unbound ASOs were then filtered and the ammonia was boiled off.The residue was purified by high pressure liquid chromatography on astrong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30μm, 2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 MNaBr in A, 0-40% of B in 60 min, flow 14 mL min-1, λ=260 nm). Theresidue was desalted by HPLC on a reverse phase column to yield thedesired ASOs in an isolated yield of 15-30% based on the initial loadingon the solid support. The ASOs were characterized by ion-pair-HPLCcoupled MS analysis with Agilent 1100 MSD system.

TABLE 34 ASO comprising a phosphodiester linked GalNAc₃-2 conjugate atthe 5′ position targeting SRB-1 Observed SEQ ID ISIS No. Sequence (5′ to3′) CalCd Mass Mass No. 661134 GalNAc ₃ -2 _(a) - _(o′) A _(do)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) 6482.2 6481.6 141G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside(e.g. cEt); “s” indicates phosphorothioate internucleoside linkages(PS); “o” indicates phosphodiester internucleoside linkages (PO); and“o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates5-methylcytosines. The structure of GalNAc₃-2_(a) is shown in Example37.

Example 42: General Method for the Preparation of ASOs Comprising aGalNAc₃-3 Conjugate at the 5′ Position Via Solid Phase Techniques(Preparation of ISIS 661166)

The synthesis for ISIS 661166 was performed using similar procedures asillustrated in Examples 39 and 41.

ISIS 661166 is a 5-10-5 MOE gapmer, wherein the 5′ position comprises aGalNAc₃-3 conjugate. The ASO was characterized by ion-pair-HPLC coupledMS analysis with Agilent 1100 MSD system.

TABLE 34a ASO comprising a GalNAc₃-3 conjugate at the 5′position via a hexylamino phosphodiester linkage targeting Malat-1 ISISCalcd Observed No. Sequence (5′ to 3′) Conjugate Mass Mass SEQ ID No.661166 5′-GalNAc₃-3_(a-o′) ^(m)C_(es)G_(es)G_(es)T_(es)G_(es) ^(m)5'-GalNAc₃-3 8992.16 8990.51 142 C_(ds)A_(ds)A_(ds)G_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(ds)A_(ds)G_(ds) G_(es)A_(es)A_(es)T_(es)T_(e)

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “s” indicates phosphorothioateinternucleoside linkages (PS); “o” indicates phosphodiesterinternucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—.Superscript “m” indicates 5-methylcytosines. The structure of“5′-GalNAc₃-3a” is shown in Example 39.

Example 43: Dose-Dependent Study of Phosphodiester Linked GalNAc₃-2 (SeeExamples 37 and 41, Bx is Adenine) at the 5′ Terminus Targeting SRB-1 InVivo

ISIS 661134 (see Example 41) comprising a phosphodiester linkedGalNAc₃-2 conjugate at the 5′ terminus was tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice. Unconjugated ISIS440762 and 651900 (GalNAc₃-1 conjugate at 3′ terminus, see Example 9)were included in the study for comparison and are described previouslyin Table 17.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 440762,651900, 661134 or with PBS treated control. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the liver SRB-1 mRNA levels usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNAlevels were determined relative to total RNA (using Ribogreen), prior tonormalization to PBS-treated control. The results below are presented asthe average percent of SRB-1 mRNA levels for each treatment group,normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀were measured using similar methods as described previously and arepresented below.

As illustrated in Table 35, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, theantisense oligonucleotides comprising the phosphodiester linkedGalNAc₃-2 conjugate at the 5′ terminus (ISIS 661134) or the GalNAc₃-1conjugate linked at the 3′ terminus (ISIS 651900) showed substantialimprovement in potency compared to the unconjugated antisenseoligonucleotide (ISIS 440762). Further, ISIS 661134, which comprises thephosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus wasequipotent compared to ISIS 651900, which comprises the GalNAc₃-1conjugate at the 3′ terminus.

TABLE 35 ASOs containing GalNAc₃-1 or GalNAc₃-2 targeting SRB-1 ISISDosage SRB-1 mRNA ED₅₀ SEQ ID No. (mg/kg) levels (% PBS) (mg/kg)Conjugate No. PBS 0 100 — — 440762 0.2 116 2.58 No 137 0.7 91 conjugate2 69 7 22 20 5 651900 0.07 95 0.26 3′ GalNAc₃-1 138 0.2 77 0.7 28 2 11 78 661134 0.07 107 0.25 5′ GalNAc₃-2 141 0.2 86 0.7 28 2 10 7 6

Structures for 3′ GalNAc₃-1 and 5′ GalNAc₃-2 were described previouslyin Examples 9 and 37.

Pharmacokinetics Analysis (PK)

The PK of the ASOs from the high dose group (7 mg/kg) was examined andevaluated in the same manner as illustrated in Example 20. Liver samplewas minced and extracted using standard protocols. The full lengthmetabolites of 661134 (5′ GalNAc₃-2) and ISIS 651900 (3′ GalNAc₃-1) wereidentified and their masses were confirmed by high resolution massspectrometry analysis. The results showed that the major metabolitedetected for the ASO comprising a phosphodiester linked GalNAc₃-2conjugate at the 5′ terminus (ISIS 661134) was ISIS 440762 (data notshown). No additional metabolites, at a detectable level, were observed.Unlike its counterpart, additional metabolites similar to those reportedpreviously in Table 23a were observed for the ASO having the GalNAc₃-1conjugate at the 3′ terminus (ISIS 651900). These results suggest thathaving the phosphodiester linked GalNAc₃-1 or GalNAc₃-2 conjugate mayimprove the PK profile of ASOs without compromising their potency.

Example 44: Effect of PO/PS Linkages on Antisense Inhibition of ASOsComprising GalNAc₃-1 Conjugate (See Example 9) at the 3′ TerminusTargeting SRB-1

ISIS 655861 and 655862 comprising a GalNAc₃-1 conjugate at the 3′terminus each targeting SRB-1 were tested in a single administrationstudy for their ability to inhibit SRB-1 in mice. The parentunconjugated compound, ISIS 353382 was included in the study forcomparison.

The ASOs are 5-10-5 MOE gapmers, wherein the gap region comprises ten2′-deoxyribonucleosides and each wing region comprises five 2′-MOEmodified nucleosides. The ASOs were prepared using similar methods asillustrated previously in Example 19 and are described Table 36, below.

TABLE 36 Modified ASOs comprising GalNAc₃-1 conjugate at the 3′ terminustargeting SRB-1 SEQ ID ISIS No. Sequence (5′ to 3′) Chemistry No. 353382G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Full PS no conjugate 143 (parent)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Full PS with 144^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -1 _(a) GalNAc ₃ -1 conjugate 655862 G_(es)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Mixed PS/PO with 144^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -1 _(a) GalNAc ₃ -1 conjugate

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “s” indicates phosphorothioateinternucleoside linkages (PS); “o” indicates phosphodiesterinternucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—.Superscript “m” indicates 5-methylcytosines. The structure of“GalNAc₃-1” is shown in Example 9.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 353382,655861, 655862 or with PBS treated control. Each treatment groupconsisted of 4 animals. Prior to the treatment as well as after the lastdose, blood was drawn from each mouse and plasma samples were analyzed.The mice were sacrificed 72 hours following the final administration todetermine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN®RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)according to standard protocols. SRB-1 mRNA levels were determinedrelative to total RNA (using Ribogreen), prior to normalization toPBS-treated control. The results below are presented as the averagepercent of SRB-1 mRNA levels for each treatment group, normalized toPBS-treated control and is denoted as “% PBS”. The ED₅₀s were measuredusing similar methods as described previously and are reported below.

As illustrated in Table 37, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner compared to PBStreated control. Indeed, the antisense oligonucleotides comprising theGalNAc₃-1 conjugate at the 3′ terminus (ISIS 655861 and 655862) showedsubstantial improvement in potency comparing to the unconjugatedantisense oligonucleotide (ISIS 353382). Further, ISIS 655862 with mixedPS/PO linkages showed an improvement in potency relative to full PS(ISIS 655861).

TABLE 37 Effect of PO/PS linkages on antisense inhibition of ASOscomprising GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1 ISISDosage SRB-1 mRNA ED₅₀ SEQ ID No. (mg/kg) levels (% PBS) (mg/kg)Chemistry No. PBS 0 100 — — 353382 3 76.65 10.4 Full PS without 143(parent) 10 52.40 conjugate 30 24.95 655861 0.5 81.22 2.2 Full PS with1.5 63.51 GalNAc₃-1 144 5 24.61 conjugate 15 14.80 655862 0.5 69.57 1.3Mixed PS/PO 144 1.5 45.78 with GalNAc₃- 5 19.70 1 conjugate 15 12.90

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Organ weights were alsoevaluated. The results demonstrated that no elevation in transaminaselevels (Table 38) or organ weights (data not shown) were observed inmice treated with ASOs compared to PBS control. Further, the ASO withmixed PS/PO linkages (ISIS 655862) showed similar transaminase levelscompared to full PS (ISIS 655861).

TABLE 38 Effect of PO/PS linkages on transaminase levels of ASOscomprising GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1 ISISDosage ALT AST No. (mg/kg) (U/L) (U/L) Chemistry SEQ ID No. PBS 0 28.565 — 353382 3 50.25 89 Full PS without 143 (parent) 10 27.5 79.3conjugate 30 27.3 97 655861 0.5 28 55.7 Full PS with 144 1.5 30 78GalNAc₃-1 5 29 63.5 15 28.8 67.8 655862 0.5 50 75.5 Mixed PS/PO with 1441.5 21.7 58.5 GalNAc₃-1 5 29.3 69 15 22 61

Example 45: Preparation of PFP Ester, Compound 110a

Compound 4 (9.5 g, 28.8 mmoles) was treated with compound 103a or 103b(38 mmoles), individually, and TMSOTf (0.5 eq.) and molecular sieves indichloromethane (200 mL), and stirred for 16 hours at room temperature.At that time, the organic layer was filtered thru celite, then washedwith sodium bicarbonate, water and brine. The organic layer was thenseparated and dried over sodium sulfate, filtered and reduced underreduced pressure. The resultant oil was purified by silica gelchromatography (2%→10% methanol/dichloromethane) to give compounds 104aand 104b in >80% yield. LCMS and proton NMR was consistent with thestructure.

Compounds 104a and 104b were treated to the same conditions as forcompounds 100a-d (Example 47), to give compounds 105a and 105b in >90%yield. LCMS and proton NMR was consistent with the structure.

Compounds 105a and 105b were treated, individually, with compound 90under the same conditions as for compounds 901a-d, to give compounds106a (80%) and 106b (20%). LCMS and proton NMR was consistent with thestructure.

Compounds 106a and 106b were treated to the same conditions as forcompounds 96a-d (Example 47), to give 107a (60%) and 107b (20%). LCMSand proton NMR was consistent with the structure.

Compounds 107a and 107b were treated to the same conditions as forcompounds 97a-d (Example 47), to give compounds 108a and 108b in 40-60%yield. LCMS and proton NMR was consistent with the structure.

Compounds 108a (60%) and 108b (40%) were treated to the same conditionsas for compounds 100a-d (Example 47), to give compounds 109a and 109bin >80% yields. LCMS and proton NMR was consistent with the structure.

Compound 109a was treated to the same conditions as for compounds 101a-d(Example 47), to give Compound 110a in 30-60% yield. LCMS and proton NMRwas consistent with the structure. Alternatively, Compound 110b can beprepared in a similar manner starting with Compound 109b.

Example 46: General Procedure for Conjugation with PFP Esters(Oligonucleotide 111); Preparation of ISIS 666881 (GalNAc₃-10)

A 5′-hexylamino modified oligonucleotide was synthesized and purifiedusing standard solid-phase oligonucleotide procedures. The 5′-hexylaminomodified oligonucleotide was dissolved in 0.1 M sodium tetraborate, pH8.5 (200 μL) and 3 equivalents of a selected PFP esterified GalNAc₃cluster dissolved in DMSO (50 μL) was added. If the PFP esterprecipitated upon addition to the ASO solution DMSO was added until allPFP ester was in solution. The reaction was complete after about 16 h ofmixing at room temperature. The resulting solution was diluted withwater to 12 mL and then spun down at 3000 rpm in a spin filter with amass cut off of 3000 Da. This process was repeated twice to remove smallmolecule impurities. The solution was then lyophilized to dryness andredissolved in concentrated aqueous ammonia and mixed at roomtemperature for 2.5 h followed by concentration in vacuo to remove mostof the ammonia. The conjugated oligonucleotide was purified and desaltedby RP-HPLC and lyophilized to provide the GalNAc₃ conjugatedoligonucleotide.

Oligonucleotide 111 is conjugated with GalNAc₃-10. The GalNAc₃ clusterportion of the conjugate group GalNAc₃-10 (GalNAc₃-10_(a)) can becombined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)— as shown in the oligonucleotide (ISIS666881) synthesized with GalNAc₃-10 below. The structure of GalNAc₃-10(GalNAc₃-10_(a)-CM-) is shown below:

Following this general procedure ISIS 666881 was prepared. 5′-hexylaminomodified oligonucleotide, ISIS 660254, was synthesized and purifiedusing standard solid-phase oligonucleotide procedures. ISIS 660254 (40mg, 5.2 μmop was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 μL)and 3 equivalents PFP ester (Compound 110a) dissolved in DMSO (50 μL)was added. The PFP ester precipitated upon addition to the ASO solutionrequiring additional DMSO (600 μL) to fully dissolve the PFP ester. Thereaction was complete after 16 h of mixing at room temperature. Thesolution was diluted with water to 12 mL total volume and spun down at3000 rpm in a spin filter with a mass cut off of 3000 Da. This processwas repeated twice to remove small molecule impurities. The solution waslyophilized to dryness and redissolved in concentrated aqueous ammoniawith mixing at room temperature for 2.5 h followed by concentration invacuo to remove most of the ammonia. The conjugated oligonucleotide waspurified and desalted by RP-HPLC and lyophilized to give ISIS 666881 in90% yield by weight (42 mg, 4.7 μmol).

GalNAc₃-10 conjugated oligonucleotide SEQ ASO Sequence (5′ to 3′)5′ group ID No. ISIS 660254 NH₂(CH₂)₆-_(o)A_(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) Hexylamine 145^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(es)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)C_(ds)T_(ds) GalNAc ₃-10 145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

Example 47: Preparation of Oligonucleotide 102 Comprising GalNAc₃-8

The triacid 90 (4 g, 14.43 mmol) was dissolved in DMF (120 mL) andN,N-Diisopropylethylamine (12.35 mL, 72 mmoles). Pentafluorophenyltrifluoroacetate (8.9 mL, 52 mmoles) was added dropwise, under argon,and the reaction was allowed to stir at room temperature for 30 minutes.Boc-diamine 91a or 91b (68.87 mmol) was added, along withN,N-Diisopropylethylamine (12.35 mL, 72 mmoles), and the reaction wasallowed to stir at room temperature for 16 hours. At that time, the DMFwas reduced by >75% under reduced pressure, and then the mixture wasdissolved in dichloromethane. The organic layer was washed with sodiumbicarbonate, water and brine. The organic layer was then separated anddried over sodium sulfate, filtered and reduced to an oil under reducedpressure. The resultant oil was purified by silica gel chromatography(2%→10% methanol/dichloromethane) to give compounds 92a and 92b in anapproximate 80% yield. LCMS and proton NMR were consistent with thestructure.

Compound 92a or 92b (6.7 mmoles) was treated with 20 mL ofdichloromethane and 20 mL of trifluoroacetic acid at room temperaturefor 16 hours. The resultant solution was evaporated and then dissolvedin methanol and treated with DOWEX-OH resin for 30 minutes. Theresultant solution was filtered and reduced to an oil under reducedpressure to give 85-90% yield of compounds 93a and 93b.

Compounds 7 or 64 (9.6 mmoles) were treated with HBTU (3.7 g, 9.6mmoles) and N,N-Diisopropylethylamine (5 mL) in DMF (20 mL) for 15minutes. To this was added either compounds 93a or 93b (3 mmoles), andallowed to stir at room temperature for 16 hours. At that time, the DMFwas reduced by >75% under reduced pressure, and then the mixture wasdissolved in dichloromethane. The organic layer was washed with sodiumbicarbonate, water and brine. The organic layer was then separated anddried over sodium sulfate, filtered and reduced to an oil under reducedpressure. The resultant oil was purified by silica gel chromatography(5%→20% methanol/dichloromethane) to give compounds 96a-d in 20-40%yield. LCMS and proton NMR was consistent with the structure.

Compounds 96a-d (0.75 mmoles), individually, were hydrogenated overRaney Nickel for 3 hours in Ethanol (75 mL). At that time, the catalystwas removed by filtration thru celite, and the ethanol removed underreduced pressure to give compounds 97a-d in 80-90% yield. LCMS andproton NMR were consistent with the structure.

Compound 23 (0.32 g, 0.53 mmoles) was treated with HBTU (0.2 g, 0.53mmoles) and N,N-Diisopropylethylamine (0.19 mL, 1.14 mmoles) in DMF (30mL) for 15 minutes. To this was added compounds 97a-d (0.38 mmoles),individually, and allowed to stir at room temperature for 16 hours. Atthat time, the DMF was reduced by >75% under reduced pressure, and thenthe mixture was dissolved in dichloromethane. The organic layer waswashed with sodium bicarbonate, water and brine. The organic layer wasthen separated and dried over sodium sulfate, filtered and reduced to anoil under reduced pressure. The resultant oil was purified by silica gelchromatography (2%→20% methanol/dichloromethane) to give compounds 98a-din 30-40% yield. LCMS and proton NMR was consistent with the structure.

Compound 99 (0.17 g, 0.76 mmoles) was treated with HBTU (0.29 g, 0.76mmoles) and N,N-Diisopropylethylamine (0.35 mL, 2.0 mmoles) in DMF (50mL) for 15 minutes. To this was added compounds 97a-d (0.51 mmoles),individually, and allowed to stir at room temperature for 16 hours. Atthat time, the DMF was reduced by >75% under reduced pressure, and thenthe mixture was dissolved in dichloromethane. The organic layer waswashed with sodium bicarbonate, water and brine. The organic layer wasthen separated and dried over sodium sulfate, filtered and reduced to anoil under reduced pressure. The resultant oil was purified by silica gelchromatography (5%→20% methanol/dichloromethane) to give compounds100a-d in 40-60% yield. LCMS and proton NMR was consistent with thestructure.

Compounds 100a-d (0.16 mmoles), individually, were hydrogenated over 10%Pd(OH)₂/C for 3 hours in methanol/ethyl acetate (1:1, 50 mL). At thattime, the catalyst was removed by filtration thru celite, and theorganics removed under reduced pressure to give compounds 101a-d in80-90% yield. LCMS and proton NMR was consistent with the structure.

Compounds 101a-d (0.15 mmoles), individually, were dissolved in DMF (15mL) and pyridine (0.016 mL, 0.2 mmoles). Pentafluorophenyltrifluoroacetate (0.034 mL, 0.2 mmoles) was added dropwise, under argon,and the reaction was allowed to stir at room temperature for 30 minutes.At that time, the DMF was reduced by >75% under reduced pressure, andthen the mixture was dissolved in dichloromethane. The organic layer waswashed with sodium bicarbonate, water and brine. The organic layer wasthen separated and dried over sodium sulfate, filtered and reduced to anoil under reduced pressure. The resultant oil was purified by silica gelchromatography (2%→5% methanol/dichloromethane) to give compounds 102a-din an approximate 80% yield. LCMS and proton NMR were consistent withthe structure.

Oligomeric Compound 102, comprising a GalNAc₃-8 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-8 (GalNAc₃-8_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In a preferred embodiment, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-8 (GalNAc₃-8_(a)-CM-) is shown below:

Example 48: Preparation of Oligonucleotide 119 Comprising GalNAc₃-7

Compound 112 was synthesized following the procedure described in theliterature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 112 (5 g, 8.6 mmol) was dissolved in 1:1 methanol/ethyl acetate(22 mL/22 mL). Palladium hydroxide on carbon (0.5 g) was added. Thereaction mixture was stirred at room temperature under hydrogen for 12h. The reaction mixture was filtered through a pad of celite and washedthe pad with 1:1 methanol/ethyl acetate. The filtrate and the washingswere combined and concentrated to dryness to yield Compound 105a(quantitative). The structure was confirmed by LCMS.

Compound 113 (1.25 g, 2.7 mmol), HBTU (3.2 g, 8.4 mmol) and DIEA (2.8mL, 16.2 mmol) were dissolved in anhydrous DMF (17 mL) and the reactionmixture was stirred at room temperature for 5 min. To this a solution ofCompound 105a (3.77 g, 8.4 mmol) in anhydrous DMF (20 mL) was added. Thereaction was stirred at room temperature for 6 h. Solvent was removedunder reduced pressure to get an oil. The residue was dissolved inCH₂Cl₂ (100 mL) and washed with aqueous saturated NaHCO₃ solution (100mL) and brine (100 mL). The organic phase was separated, dried (Na₂SO₄),filtered and evaporated. The residue was purified by silica gel columnchromatography and eluted with 10 to 20% MeOH in dichloromethane toyield Compound 114 (1.45 g, 30%). The structure was confirmed by LCMSand ¹H NMR analysis.

Compound 114 (1.43 g, 0.8 mmol) was dissolved in 1:1 methanol/ethylacetate (4 mL/4 mL). Palladium on carbon (wet, 0.14 g) was added. Thereaction mixture was flushed with hydrogen and stirred at roomtemperature under hydrogen for 12 h. The reaction mixture was filteredthrough a pad of celite. The celite pad was washed with methanol/ethylacetate (1:1). The filtrate and the washings were combined together andevaporated under reduced pressure to yield Compound 115 (quantitative).The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 83a (0.17 g, 0.75 mmol), HBTU (0.31 g, 0.83 mmol) and DIEA(0.26 mL, 1.5 mmol) were dissolved in anhydrous DMF (5 mL) and thereaction mixture was stirred at room temperature for 5 min. To this asolution of Compound 115 (1.22 g, 0.75 mmol) in anhydrous DMF was addedand the reaction was stirred at room temperature for 6 h. The solventwas removed under reduced pressure and the residue was dissolved inCH₂Cl₂. The organic layer was washed aqueous saturated NaHCO₃ solutionand brine and dried over anhydrous Na₂SO₄ and filtered. The organiclayer was concentrated to dryness and the residue obtained was purifiedby silica gel column chromatography and eluted with 3 to 15% MeOH indichloromethane to yield Compound 116 (0.84 g, 61%). The structure wasconfirmed by LC MS and ¹H NMR analysis.

Compound 116 (0.74 g, 0.4 mmol) was dissolved in 1:1 methanol/ethylacetate (5 mL/5 mL). Palladium on carbon (wet, 0.074 g) was added. Thereaction mixture was flushed with hydrogen and stirred at roomtemperature under hydrogen for 12 h. The reaction mixture was filteredthrough a pad of celite. The celite pad was washed with methanol/ethylacetate (1:1). The filtrate and the washings were combined together andevaporated under reduced pressure to yield compound 117 (0.73 g, 98%).The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 117 (0.63 g, 0.36 mmol) was dissolved in anhydrous DMF (3 mL).To this solution N,N-Diisopropylethylamine (70 μL, 0.4 mmol) andpentafluorophenyl trifluoroacetate (72 μL, 0.42 mmol) were added. Thereaction mixture was stirred at room temperature for 12 h and pouredinto a aqueous saturated NaHCO₃ solution. The mixture was extracted withdichloromethane, washed with brine and dried over anhydrous Na₂SO₄. Thedichloromethane solution was concentrated to dryness and purified withsilica gel column chromatography and eluted with 5 to 10% MeOH indichloromethane to yield compound 118 (0.51 g, 79%). The structure wasconfirmed by LCMS and ¹H and ¹H and ¹⁹F NMR.

Oligomeric Compound 119, comprising a GalNAc₃-7 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-7 (GalNAc₃-7_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-7 (GalNAc₃-7_(a)-CM-) is shown below:

Example 49: Preparation of Oligonucleotide 132 Comprising GalNAc₃-5

Compound 120 (14.01 g, 40 mmol) and HBTU (14.06 g, 37 mmol) weredissolved in anhydrous DMF (80 mL). Triethylamine (11.2 mL, 80.35 mmol)was added and stirred for 5 min. The reaction mixture was cooled in anice bath and a solution of compound 121 (10 g, mmol) in anhydrous DMF(20 mL) was added. Additional triethylamine (4.5 mL, 32.28 mmol) wasadded and the reaction mixture was stirred for 18 h under an argonatmosphere. The reaction was monitored by TLC (ethyl acetate:hexane;1:1; Rf=0.47). The solvent was removed under reduced pressure. Theresidue was taken up in EtOAc (300 mL) and washed with 1M NaHSO₄ (3×150mL), aqueous saturated NaHCO₃ solution (3×150 mL) and brine (2×100 mL).Organic layer was dried with Na₂SO₄. Drying agent was removed byfiltration and organic layer was concentrated by rotary evaporation.Crude mixture was purified by silica gel column chromatography andeluted by using 35-50% EtOAc in hexane to yield a compound 122 (15.50 g,78.13%). The structure was confirmed by LCMS and ¹H NMR analysis. Massm/z 589.3 [M+H]⁺.

A solution of LiOH (92.15 mmol) in water (20 mL) and THF (10 mL) wasadded to a cooled solution of Compound 122 (7.75 g, 13.16 mmol)dissolved in methanol (15 mL). The reaction mixture was stirred at roomtemperature for 45 min. and monitored by TLC (EtOAc:hexane; 1:1). Thereaction mixture was concentrated to half the volume under reducedpressure. The remaining solution was cooled an ice bath and neutralizedby adding concentrated HCl. The reaction mixture was diluted, extractedwith EtOAc (120 mL) and washed with brine (100 mL). An emulsion formedand cleared upon standing overnight. The organic layer was separateddried (Na₂SO₄), filtered and evaporated to yield Compound 123 (8.42 g).Residual salt is the likely cause of excess mass. LCMS is consistentwith structure. Product was used without any further purification. M.W.cal: 574.36; M.W. fd: 575.3 [M+H]⁺.

Compound 126 was synthesized following the procedure described in theliterature (J. Am. Chem. Soc. 2011, 133, 958-963).

Compound 123 (7.419 g, 12.91 mmol), HOBt (3.49 g, 25.82 mmol) andcompound 126 (6.33 g, 16.14 mmol) were dissolved in and DMF (40 mL) andthe resulting reaction mixture was cooled in an ice bath. To thisN,N-Diisopropylethylamine (4.42 mL, 25.82 mmol), PyBop (8.7 g, 16.7mmol) followed by Bop coupling reagent (1.17 g, 2.66 mmol) were addedunder an argon atmosphere. The ice bath was removed and the solution wasallowed to warm to room temperature. The reaction was completed after 1h as determined by TLC (DCM:MeOH:AA; 89:10:1). The reaction mixture wasconcentrated under reduced pressure. The residue was dissolved in EtOAc(200 mL) and washed with 1 M NaHSO₄ (3×100 mL), aqueous saturated NaHCO₃(3×100 mL) and brine (2×100 mL). The organic phase separated dried(Na₂SO₄), filtered and concentrated. The residue was purified by silicagel column chromatography with a gradient of 50% hexanes/EtOAC to 100%EtOAc to yield Compound 127 (9.4 g) as a white foam. LCMS and ¹H NMRwere consistent with structure. Mass m/z 778.4 [M+H]⁺.

Trifluoroacetic acid (12 mL) was added to a solution of compound 127(1.57 g, 2.02 mmol) in dichloromethane (12 mL) and stirred at roomtemperature for 1 h. The reaction mixture was co-evaporated with toluene(30 mL) under reduced pressure to dryness. The residue obtained wasco-evaporated twice with acetonitrile (30 mL) and toluene (40 mL) toyield Compound 128 (1.67 g) as trifluoro acetate salt and used for nextstep without further purification. LCMS and ¹H NMR were consistent withstructure. Mass m/z 478.2 [M+H]⁺.

Compound 7 (0.43 g, 0.963 mmol), HATU (0.35 g, 0.91 mmol), and HOAt(0.035 g, 0.26 mmol) were combined together and dried for 4 h over P₂O₅under reduced pressure in a round bottom flask and then dissolved inanhydrous DMF (1 mL) and stirred for 5 min. To this a solution ofcompound 128 (0.20 g, 0.26 mmol) in anhydrous DMF (0.2 mL) andN,N-Diisopropylethylamine (0.2 mL) was added. The reaction mixture wasstirred at room temperature under an argon atmosphere. The reaction wascomplete after 30 min as determined by LCMS and TLC (7% MeOH/DCM). Thereaction mixture was concentrated under reduced pressure. The residuewas dissolved in DCM (30 mL) and washed with 1 M NaHSO₄ (3×20 mL),aqueous saturated NaHCO₃ (3×20 mL) and brine (3×20 mL). The organicphase was separated, dried over Na₂SO₄, filtered and concentrated. Theresidue was purified by silica gel column chromatography using 5-15%MeOH in dichloromethane to yield Compound 129 (96.6 mg). LC MS and NMRare consistent with structure. Mass m/z 883.4 [M+2H]⁺.

Compound 129 (0.09 g, 0.051 mmol) was dissolved in methanol (5 mL) in 20mL scintillation vial. To this was added a small amount of 10% Pd/C(0.015 mg) and the reaction vessel was flushed with H₂ gas. The reactionmixture was stirred at room temperature under H₂ atmosphere for 18 h.The reaction mixture was filtered through a pad of Celite and the Celitepad was washed with methanol. The filtrate washings were pooled togetherand concentrated under reduced pressure to yield Compound 130 (0.08 g).LCMS and NMR were consistent with structure. The product was usedwithout further purification. Mass m/z 838.3 [M+2H]⁺.

To a 10 mL pointed round bottom flask were added compound 130 (75.8 mg,0.046 mmol), 0.37 M pyridine/DMF (200 μL) and a stir bar. To thissolution was added 0.7 M pentafluorophenyl trifluoroacetate/DMF (100 μL)drop wise with stirring. The reaction was completed after 1 h asdetermined by LC MS. The solvent was removed under reduced pressure andthe residue was dissolved in CHCl₃ (˜10 mL). The organic layer waspartitioned against NaHSO₄ (1 M, 10 mL), aqueous saturated NaHCO₃ (10mL) and brine (10 mL) three times each. The organic phase separated anddried over Na₂SO₄, filtered and concentrated to yield Compound 131 (77.7mg). LCMS is consistent with structure. Used without furtherpurification. Mass m/z 921.3 [M+2H]⁺.

Oligomeric Compound 132, comprising a GalNAc₃-5 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-5 (GalNAc₃-5_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-5 (GalNAc₃-5_(a)-CM-) is shown below:

Example 50: Preparation of Oligonucleotide 144 Comprising GalNAc₄-11

Synthesis of Compound 134. To a Merrifield flask was added aminomethylVIMAD resin (2.5 g, 450 μmol/g) that was washed with acetonitrile,dimethylformamide, dichloromethane and acetonitrile. The resin wasswelled in acetonitrile (4 mL). Compound 133 was pre-activated in a 100mL round bottom flask by adding 20 (1.0 mmol, 0.747 g), TBTU (1.0 mmol,0.321 g), acetonitrile (5 mL) and DIEA (3.0 mmol, 0.5 mL). This solutionwas allowed to stir for 5 min and was then added to the Merrifield flaskwith shaking. The suspension was allowed to shake for 3 h. The reactionmixture was drained and the resin was washed with acetonitrile, DMF andDCM. New resin loading was quantitated by measuring the absorbance ofthe DMT cation at 500 nm (extinction coefficient=76000) in DCM anddetermined to be 238 μmol/g. The resin was capped by suspending in anacetic anhydride solution for ten minutes three times.

The solid support bound compound 141 was synthesized using iterativeFmoc-based solid phase peptide synthesis methods. A small amount ofsolid support was withdrawn and suspended in aqueous ammonia (28-30 wt%) for 6 h. The cleaved compound was analyzed by LC-MS and the observedmass was consistent with structure. Mass m/z 1063.8 [M+2H]⁺.

The solid support bound compound 142 was synthesized using solid phasepeptide synthesis methods.

The solid support bound compound 143 was synthesized using standardsolid phase synthesis on a DNA synthesizer.

The solid support bound compound 143 was suspended in aqueous ammonia(28-30 wt %) and heated at 55° C. for 16 h. The solution was cooled andthe solid support was filtered. The filtrate was concentrated and theresidue dissolved in water and purified by HPLC on a strong anionexchange column. The fractions containing full length compound 144 werepooled together and desalted. The resulting GalNAc₄-11 conjugatedoligomeric compound was analyzed by LC-MS and the observed mass wasconsistent with structure.

The GalNAc₄ cluster portion of the conjugate group GalNAc₄-11(GalNAc₄-11_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₄-11 (GalNAc₄-11_(a)-CM) is shown below:

Example 51: Preparation of Oligonucleotide 155 Comprising GalNAc₃-6

Compound 146 was synthesized as described in the literature (AnalyticalBiochemistry 1995, 229, 54-60).

Compound 4 (15 g, 45.55 mmol) and compound 35b (14.3 grams, 57 mmol)were dissolved in CH₂Cl₂ (200 ml). Activated molecular sieves (4 Å, 2 g,powdered) were added, and the reaction was allowed to stir for 30minutes under nitrogen atmosphere. TMS-OTf was added (4.1 ml, 22.77mmol) and the reaction was allowed to stir at room temp overnight. Uponcompletion, the reaction was quenched by pouring into solution ofsaturated aqueous NaHCO₃ (500 ml) and crushed ice (˜150 g). The organiclayer was separated, washed with brine, dried over MgSO₄, filtered, andwas concentrated to an orange oil under reduced pressure. The crudematerial was purified by silica gel column chromatography and elutedwith 2-10% MeOH in CH₂Cl₂ to yield Compound 112 (16.53 g, 63%). LCMS and¹H NMR were consistent with the expected compound.

Compound 112 (4.27 g, 7.35 mmol) was dissolved in 1:1 MeOH/EtOAc (40ml). The reaction mixture was purged by bubbling a stream of argonthrough the solution for 15 minutes. Pearlman's catalyst (palladiumhydroxide on carbon, 400 mg) was added, and hydrogen gas was bubbledthrough the solution for 30 minutes. Upon completion (TLC 10% MeOH inCH₂Cl₂, and LCMS), the catalyst was removed by filtration through a padof celite. The filtrate was concentrated by rotary evaporation, and wasdried briefly under high vacuum to yield Compound 105a (3.28 g). LCMSand 1H NMR were consistent with desired product.

Compound 147 (2.31 g, 11 mmol) was dissolved in anhydrous DMF (100 mL).N,N-Diisopropylethylamine (DIEA, 3.9 mL, 22 mmol) was added, followed byHBTU (4 g, 10.5 mmol). The reaction mixture was allowed to stir for ˜15minutes under nitrogen. To this a solution of compound 105a (3.3 g, 7.4mmol) in dry DMF was added and stirred for 2 h under nitrogenatmosphere. The reaction was diluted with EtOAc and washed withsaturated aqueous NaHCO₃ and brine. The organics phase was separated,dried (MgSO₄), filtered, and concentrated to an orange syrup. The crudematerial was purified by column chromatography 2-5% MeOH in CH₂Cl₂ toyield Compound 148 (3.44 g, 73%). LCMS and ¹H NMR were consistent withthe expected product.

Compound 148 (3.3 g, 5.2 mmol) was dissolved in 1:1 MeOH/EtOAc (75 ml).The reaction mixture was purged by bubbling a stream of argon throughthe solution for 15 minutes. Pearlman's catalyst (palladium hydroxide oncarbon) was added (350 mg). Hydrogen gas was bubbled through thesolution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, andLCMS), the catalyst was removed by filtration through a pad of celite.The filtrate was concentrated by rotary evaporation, and was driedbriefly under high vacuum to yield Compound 149 (2.6 g). LCMS wasconsistent with desired product. The residue was dissolved in dry DMF(10 ml) was used immediately in the next step.

Compound 146 (0.68 g, 1.73 mmol) was dissolved in dry DMF (20 ml). Tothis DIEA (450 μL, 2.6 mmol, 1.5 eq.) and HBTU (1.96 g, 0.5.2 mmol) wereadded. The reaction mixture was allowed to stir for 15 minutes at roomtemperature under nitrogen. A solution of compound 149 (2.6 g) inanhydrous DMF (10 mL) was added. The pH of the reaction was adjusted topH=9-10 by addition of DIEA (if necessary). The reaction was allowed tostir at room temperature under nitrogen for 2 h. Upon completion thereaction was diluted with EtOAc (100 mL), and washed with aqueoussaturated aqueous NaHCO₃, followed by brine. The organic phase wasseparated, dried over MgSO₄, filtered, and concentrated. The residue waspurified by silica gel column chromatography and eluted with 2-10% MeOHin CH₂Cl₂ to yield Compound 150 (0.62 g, 20%). LCMS and ¹H NMR wereconsistent with the desired product.

Compound 150 (0.62 g) was dissolved in 1:1 MeOH/EtOAc (5 L). Thereaction mixture was purged by bubbling a stream of argon through thesolution for 15 minutes. Pearlman's catalyst (palladium hydroxide oncarbon) was added (60 mg). Hydrogen gas was bubbled through the solutionfor 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), thecatalyst was removed by filtration (syringe tip Teflon filter, 0.45 μm).The filtrate was concentrated by rotary evaporation, and was driedbriefly under high vacuum to yield Compound 151 (0.57 g). The LCMS wasconsistent with the desired product. The product was dissolved in 4 mLdry DMF and was used immediately in the next step.

Compound 83a (0.11 g, 0.33 mmol) was dissolved in anhydrous DMF (5 mL)and N,N-Diisopropylethylamine (75 μL, 1 mmol) and PFP-TFA (90 μL, 0.76mmol) were added. The reaction mixture turned magenta upon contact, andgradually turned orange over the next 30 minutes. Progress of reactionwas monitored by TLC and LCMS. Upon completion (formation of the PFPester), a solution of compound 151 (0.57 g, 0.33 mmol) in DMF was added.The pH of the reaction was adjusted to pH=9-10 by addition ofN,N-Diisopropylethylamine (if necessary). The reaction mixture wasstirred under nitrogen for ˜30 min. Upon completion, the majority of thesolvent was removed under reduced pressure. The residue was diluted withCH₂Cl₂ and washed with aqueous saturated NaHCO₃, followed by brine. Theorganic phase separated, dried over MgSO₄, filtered, and concentrated toan orange syrup. The residue was purified by silica gel columnchromatography (2-10% MeOH in CH₂Cl₂) to yield Compound 152 (0.35 g,55%). LCMS and ¹H NMR were consistent with the desired product.

Compound 152 (0.35 g, 0.182 mmol) was dissolved in 1:1 MeOH/EtOAc (10mL). The reaction mixture was purged by bubbling a stream of argon thruthe solution for 15 minutes. Pearlman's catalyst (palladium hydroxide oncarbon) was added (35 mg). Hydrogen gas was bubbled thru the solutionfor 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), thecatalyst was removed by filtration (syringe-tip Teflon filter, 0.45 μm).The filtrate was concentrated by rotary evaporation, and was driedbriefly under high vacuum to yield Compound 153 (0.33 g, quantitative).The LCMS was consistent with desired product.

Compound 153 (0.33 g, 0.18 mmol) was dissolved in anhydrous DMF (5 mL)with stirring under nitrogen. To this N,N-Diisopropylethylamine (65 μL,0.37 mmol) and PFP-TFA (35 μL, 0.28 mmol) were added. The reactionmixture was stirred under nitrogen for ˜30 min. The reaction mixtureturned magenta upon contact, and gradually turned orange. The pH of thereaction mixture was maintained at pH=9-10 by adding moreN,-Diisopropylethylamine. The progress of the reaction was monitored byTLC and LCMS. Upon completion, the majority of the solvent was removedunder reduced pressure. The residue was diluted with CH₂Cl₂ (50 mL), andwashed with saturated aqueous NaHCO₃, followed by brine. The organiclayer was dried over MgSO₄, filtered, and concentrated to an orangesyrup. The residue was purified by column chromatography and eluted with2-10% MeOH in CH₂Cl₂ to yield Compound 154 (0.29 g, 79%). LCMS and ¹HNMR were consistent with the desired product.

Oligomeric Compound 155, comprising a GalNAc₃-6 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-6 (GalNAc₃-6_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-6 (GalNAc₃-6_(a)-CM-) is shown below:

Example 52: Preparation of Oligonucleotide 160 Comprising GalNAc₃-9

Compound 156 was synthesized following the procedure described in theliterature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 156, (18.60 g, 29.28 mmol) was dissolved in methanol (200 mL).Palladium on carbon (6.15 g, 10 wt %, loading (dry basis), matrix carbonpowder, wet) was added. The reaction mixture was stirred at roomtemperature under hydrogen for 18 h. The reaction mixture was filteredthrough a pad of celite and the celite pad was washed thoroughly withmethanol. The combined filtrate was washed and concentrated to dryness.The residue was purified by silica gel column chromatography and elutedwith 5-10% methanol in dichloromethane to yield Compound 157 (14.26 g,89%). Mass m/z 544.1 [M−H]⁻.

Compound 157 (5 g, 9.17 mmol) was dissolved in anhydrous DMF (30 mL).HBTU (3.65 g, 9.61 mmol) and N,N-Diisopropylethylamine (13.73 mL, 78.81mmol) were added and the reaction mixture was stirred at roomtemperature for 5 minutes. To this a solution of compound 47 (2.96 g,7.04 mmol) was added. The reaction was stirred at room temperature for 8h. The reaction mixture was poured into a saturated NaHCO₃ aqueoussolution. The mixture was extracted with ethyl acetate and the organiclayer was washed with brine and dried (Na₂SO₄), filtered and evaporated.The residue obtained was purified by silica gel column chromatographyand eluted with 50% ethyl acetate in hexane to yield compound 158 (8.25g, 73.3%). The structure was confirmed by MS and ¹H NMR analysis.

Compound 158 (7.2 g, 7.61 mmol) was dried over P₂O₅ under reducedpressure. The dried compound was dissolved in anhydrous DMF (50 mL). Tothis 1H-tetrazole (0.43 g, 6.09 mmol) and N-methylimidazole (0.3 mL,3.81 mmol) and 2-cyanoethyl-N,N,N′,N′-tetraisopropyl phosphorodiamidite(3.65 mL, 11.50 mmol) were added. The reaction mixture was stirred tunder an argon atmosphere for 4 h. The reaction mixture was diluted withethyl acetate (200 mL). The reaction mixture was washed with saturatedNaHCO₃ and brine. The organic phase was separated, dried (Na₂SO₄),filtered and evaporated. The residue was purified by silica gel columnchromatography and eluted with 50-90% ethyl acetate in hexane to yieldCompound 159 (7.82 g, 80.5%). The structure was confirmed by LCMS and³¹P NMR analysis.

Oligomeric Compound 160, comprising a GalNAc₃-9 conjugate group, wasprepared using standard oligonucleotide synthesis procedures. Threeunits of compound 159 were coupled to the solid support, followed bynucleotide phosphoramidites. Treatment of the protected oligomericcompound with aqueous ammonia yielded compound 160. The GalNAc₃ clusterportion of the conjugate group GalNAc₃-9 (GalNAc₃-9_(a)) can be combinedwith any cleavable moiety to provide a variety of conjugate groups. Incertain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-9(GalNAc₃-9_(a)-CM) is shown below:

Example 53: Alternate Procedure for Preparation of Compound 18(GalNAc₃-1a and GalNAc₃-3a)

Lactone 161 was reacted with diamino propane (3-5 eq) or Mono-Bocprotected diamino propane (1 eq) to provide alcohol 162a or 162b. Whenunprotected propanediamine was used for the above reaction, the excessdiamine was removed by evaporation under high vacuum and the free aminogroup in 162a was protected using CbzCl to provide 162b as a white solidafter purification by column chromatography. Alcohol 162b was furtherreacted with compound 4 in the presence of TMSOTf to provide 163a whichwas converted to 163b by removal of the Cbz group using catalytichydrogenation. The pentafluorophenyl (PFP) ester 164 was prepared byreacting triacid 113 (see Example 48) with PFPTFA (3.5 eq) and pyridine(3.5 eq) in DMF (0.1 to 0.5 M). The triester 164 was directly reactedwith the amine 163b (3-4 eq) and DIPEA (3-4 eq) to provide Compound 18.The above method greatly facilitates purification of intermediates andminimizes the formation of byproducts which are formed using theprocedure described in Example 4.

Example 54: Alternate Procedure for Preparation of Compound 18(GalNAc₃-1a and GalNAc₃-3a)

The triPFP ester 164 was prepared from acid 113 using the procedureoutlined in example 53 above and reacted with mono-Boc protected diamineto provide 165 in essentially quantitative yield. The Boc groups wereremoved with hydrochloric acid or trifluoroacetic acid to provide thetriamine which was reacted with the PFP activated acid 166 in thepresence of a suitable base such as DIPEA to provide Compound 18.

The PFP protected Gal-NAc acid 166 was prepared from the correspondingacid by treatment with PFPTFA (1-1.2 eq) and pyridine (1-1.2 eq) in DMF.The precursor acid in turn was prepared from the corresponding alcoholby oxidation using TEMPO (0.2 eq) and BAIB in acetonitrile and water.The precursor alcohol was prepared from sugar intermediate 4 by reactionwith 1,6-hexanediol (or 1,5-pentanediol or other diol for other nvalues) (2-4 eq) and TMSOTf using conditions described previously inexample 47.

Example 55: Dose-Dependent Study of Oligonucleotides Comprising Either a3′ or 5′-Conjugate Group (Comparison of GalNAc₃-1, 3, 8 and 9) TargetingSRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 wasincluded as a standard. Each of the various GalNAc₃ conjugate groups wasattached at either the 3′ or 5′ terminus of the respectiveoligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside(cleavable moiety).

TABLE 39 Modified ASO targeting SRB-1 SEQ ID ASO Sequence (5′ to 3′)Motif Conjugate No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5none 143 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 655861 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃ -1 144 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo)A _(do′) -GalNAc ₃ -1 _(a) ISIS 664078 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃ -9 144 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo)A _(do′) -GalNAc ₃ -9 _(a) ISIS 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)5/10/5 GalNAc ₃ -3 145 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 665001GalNAc ₃ -8 _(a) - _(o′) A _(do) 5/10/5 GalNAc ₃ -8 145 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. Thestructure of GalNAc₃-9 was shown previously in Example 52. The structureof GalNAc₃-3 was shown previously in Example 39. The structure ofGalNAc₃-8 was shown previously in Example 47.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 353382,655861, 664078, 661161, 665001 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the liver SRB-1 mRNA levels usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. The resultsbelow are presented as the average percent of SRB-1 mRNA levels for eachtreatment group, normalized to the saline control.

As illustrated in Table 40, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, theantisense oligonucleotides comprising the phosphodiester linkedGalNAc₃-1 and GalNAc₃-9 conjugates at the 3′ terminus (ISIS 655861 andISIS 664078) and the GalNAc₃-3 and GalNAc₃-8 conjugates linked at the 5′terminus (ISIS 661161 and ISIS 665001) showed substantial improvement inpotency compared to the unconjugated antisense oligonucleotide (ISIS353382). Furthermore, ISIS 664078, comprising a GalNAc₃-9 conjugate atthe 3′ terminus was essentially equipotent compared to ISIS 655861,which comprises a GalNAc₃-1 conjugate at the 3′ terminus. The 5′conjugated antisense oligonucleotides, ISIS 661161 and ISIS 665001,comprising a GalNAc₃-3 or GalNAc₃-9, respectively, had increased potencycompared to the 3′ conjugated antisense oligonucleotides (ISIS 655861and ISIS 664078).

TABLE 40 ASOs containing GalNAc₃-1, 3, 8 or 9 targeting SRB-1 DosageSRB-1 mRNA ISIS No. (mg/kg) (% Saline) Conjugate Saline n/a 100 353382 388 none 10 68 30 36 655861 0.5 98 GalNac₃-1 (3′) 1.5 76 5 31 15 20664078 0.5 88 GalNac₃-9 (3′) 1.5 85 5 46 15 20 GalNac₃-3 (5′) 661161 0.592 1.5 59 5 19 15 11 GalNac₃-8 (5′) 665001 0.5 100 1.5 73 5 29 15 13

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group. ALTs, ASTs, total bilirubinand BUN values are shown in the table below.

TABLE 41 ISIS Dosage Total No. mg/kg ALT AST Bilirubin BUN ConjugateSaline 24 59 0.1 37.52 353382 3 21 66 0.2 34.65 none 10 22 54 0.2 34.230 22 49 0.2 33.72 655861 0.5 25 62 0.2 30.65 GalNac₃-1 (3′) 1.5 23 480.2 30.97 5 28 49 0.1 32.92 15 40 97 0.1 31.62 664078 0.5 40 74 0.1 35.3GalNac₃-9 (3′) 1.5 47 104 0.1 32.75 5 20 43 0.1 30.62 15 38 92 0.1 26.2661161 0.5 101 162 0.1 34.17 GalNac₃-3 (5′) 1.5 g 42 100 0.1 33.37 5 g23 99 0.1 34.97 15 53 83 0.1 34.8 665001 0.5 28 54 0.1 31.32 GalNac₃-8(5′) 1.5 42 75 0.1 32.32 5 24 42 0.1 31.85 15 32 67 0.1 31.

Example 56: Dose-Dependent Study of Oligonucleotides Comprising Either a3′ or 5′-Conjugate Group (Comparison of GalNAc₃-1, 2, 3, 5, 6, 7 and 10)Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 wasincluded as a standard. Each of the various GalNAc₃ conjugate groups wasattached at the 5′ terminus of the respective oligonucleotide by aphosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety)except for ISIS 655861 which had the GalNAc₃ conjugate group attached atthe 3′ terminus.

TABLE 42 Modified ASO targeting SRB-1 SEQ ASO Sequence (5′ to 3′) MotifConjugate ID No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5no conjugate 143 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 655861 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃ -1 144 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo)A _(do′) -GalNAc ₃ -1 _(a) ISIS 664507 GalNAc ₃ -2 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5GalNAc ₃ -2 145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 661161GalNAc ₃ -3 _(a) - _(o′) A _(do) 5/10/5 GalNAc ₃ -3 145 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666224 GalNAc ₃ -5 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -5145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666961 GalNAc ₃ -6 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -6145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666981 GalNAc ₃ -7 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -7145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5GalNAc ₃ -10 145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. Thestructure of GalNAc₃-2_(a) was shown previously in Example 37. Thestructure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-5_(a) was shown previously in Example 49. Thestructure of GalNAc₃-6_(a) was shown previously in Example 51. Thestructure of GalNAc₃-7_(a) was shown previously in Example 48. Thestructure of GalNAc₃-10_(a) was shown previously in Example 46.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 353382,655861, 664507, 661161, 666224, 666961, 666981, 666881 or with saline.Each treatment group consisted of 4 animals. The mice were sacrificed 72hours following the final administration to determine the liver SRB-1mRNA levels using real-time PCR and RIBOGREEN® RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.) according to standardprotocols. The results below are presented as the average percent ofSRB-1 mRNA levels for each treatment group, normalized to the salinecontrol.

As illustrated in Table 43, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, theconjugated antisense oligonucleotides showed substantial improvement inpotency compared to the unconjugated antisense oligonucleotide (ISIS353382). The 5′ conjugated antisense oligonucleotides showed a slightincrease in potency compared to the 3′ conjugated antisenseoligonucleotide.

TABLE 43 Dosage SRB-1 mRNA ISIS No. (mg/kg) (% Saline) Conjugate Salinen/a 100.0 353382 3 96.0 none 10 73.1 30 36.1 655861 0.5 99.4 GalNac₃-1(3′) 1.5 81.2 5 33.9 15 15.2 664507 0.5 102.0 GalNac₃-2 (5′) 1.5 73.2 531.3 15 10.8 661161 0.5 90.7 GalNac₃-3 (5′) 1.5 67.6 5 24.3 15 11.5666224 0.5 96.1 GalNac₃-5 (5′) 1.5 61.6 5 25.6 15 11.7 666961 0.5 85.5GalNAc₃-6 (5′) 1.5 56.3 5 34.2 15 13.1 666981 0.5 84.7 GalNAc₃-7 (5′)1.5 59.9 5 24.9 15 8.5 666881 0.5 100.0  GalNAc₃-10 (5′) 1.5 65.8 5 26.015 13.0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group. ALTs, ASTs, total bilirubinand BUN values are shown in Table 44 below.

TABLE 44 Dosage Total ISIS No. mg/kg ALT AST Bilirubin BUN ConjugateSaline 26 57 0.2 27 353382 3 25 92 0.2 27 none 10 23 40 0.2 25 30 29 540.1 28 655861 0.5 25 71 0.2 34 GalNac₃-1 (3′) 1.5 28 60 0.2 26 5 26 630.2 28 15 25 61 0.2 28 664507 0.5 25 62 0.2 25 GalNac₃-2 (5′) 1.5 24 490.2 26 5 21 50 0.2 26 15 59 84 0.1 22 661161 0.5 20 42 0.2 29 GalNac₃-3(5′) 1.5 g 37 74 0.2 25   5 g 28 61 0.2 29 15 21 41 0.2 25 666224 0.5 3448 0.2 21 GalNac₃-5 (5′) 1.5 23 46 0.2 26 5 24 47 0.2 23 15 32 49 0.1 26666961 0.5 17 63 0.2 26 GalNAc₃-6 (5′) 1.5 23 68 0.2 26 5 25 66 0.2 2615 29 107 0.2 28 666981 0.5 24 48 0.2 26 GalNAc₃-7 (5′) 1.5 30 55 0.2 245 46 74 0.1 24 15 29 58 0.1 26 666881 0.5 20 65 0.2 27 GalNAc₃-10 (5′)1.5 23 59 0.2 24 5 45 70 0.2 26 15 21 57 0.2 24

Example 57: Duration of Action Study of Oligonucleotides Comprising a3′-Conjugate Group Targeting ApoC III In Vivo

Mice were injected once with the doses indicated below and monitoredover the course of 42 days for ApoC-III and plasma triglycerides (PlasmaTG) levels. The study was performed using 3 transgenic mice that expresshuman APOC-III in each group.

TABLE 45 Modified ASO targeting ApoC III SEQ ID ASO Sequence (5′ to 3′)Linkages No. ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) PS 135 304801 ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ISISA_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds) PS 136 647535 A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′)-GalNAc ₃-1 _(a) ISISA_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds) PO/PS 136 647536 A_(ds)G_(ds)^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(eo) A _(do′)-GalNAc ₃-1 _(a)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1 a was shown previously in Example 9.

TABLE 46 ApoC III mRNA (% Saline on Day 1) and Plasma TG Levels (%Saline on Day 1) Day Day Day Day Day ASO Dose Target 3 7 14 35 42 Saline 0 mg/kg ApoC-III 98 100 100 95 116 ISIS 304801 30 mg/kg ApoC-III 28 3041 65 74 ISIS 647535 10 mg/kg ApoC-III 16 19 25 74 94 ISIS 647536 10mg/kg ApoC-III 18 16 17 35 51 Saline  0 mg/kg Plasma 121 130 123 105 109TG ISIS 304801 30 mg/kg Plasma 34 37 50 69 69 TG ISIS 647535 10 mg/kgPlasma 18 14 24 18 71 TG ISIS 647536 10 mg/kg Plasma 21 19 15 32 35 TG

As can be seen in the table above the duration of action increased withaddition of the 3′-conjugate group compared to the unconjugatedoligonucleotide. There was a further increase in the duration of actionfor the conjugated mixed PO/PS oligonucleotide 647536 as compared to theconjugated full PS oligonucleotide 647535.

Example 58: Dose-Dependent Study of Oligonucleotides Comprising a3′-Conjugate Group (Comparison of GalNAc₃-1 and GalNAc₄-11) TargetingSRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 440762 wasincluded as an unconjugated standard. Each of the conjugate groups wereattached at the 3′ terminus of the respective oligonucleotide by aphosphodiester linked 2′-deoxyadenosine nucleoside cleavable moiety.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. Thestructure of GalNAc₃-11_(a) was shown previously in Example 50.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 440762,651900, 663748 or with saline. Each treatment group consisted of 4animals. The mice were sacrificed 72 hours following the finaladministration to determine the liver SRB-1 mRNA levels using real-timePCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.Eugene, Oreg.) according to standard protocols. The results below arepresented as the average percent of SRB-1 mRNA levels for each treatmentgroup, normalized to the saline control.

As illustrated in Table 47, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. The antisenseoligonucleotides comprising the phosphodiester linked GalNAc₃-1 andGalNAc₄-11 conjugates at the 3′ terminus (ISIS 651900 and ISIS 663748)showed substantial improvement in potency compared to the unconjugatedantisense oligonucleotide (ISIS 440762). The two conjugatedoligonucleotides, GalNAc₃-1 and GalNAc₄-11, were equipotent.

TABLE 47 Modified ASO targeting SRB-1 % Saline SEQ ID ASO Sequence(5′ to 3′) Dose mg/kg control No. Saline 100 ISIS 440762 T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.673.45 137 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) 2 59.66 6 23.50 ISIS 651900T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)0.2 62.75 138 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do′)-GalNAc ₃-1 _(a)0.6 29.14 2 8.61 6 5.62 ISIS 663748 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.2 63.99 138 ^(m)C_(ds)T_(ds)T_(ks)^(m)C_(ko) A _(do′)-GalNAc ₄-11 _(a) 0.6 33.53 2 7.58 6 5.52

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside; “d”indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioateinternucleoside linkage (PS); “o” indicates a phosphodiesterinternucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—.Conjugate groups are in bold.

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group. ALTs, ASTs, total bilirubinand BUN values are shown in Table 48 below.

TABLE 48 Dosage Total ISIS No. mg/kg ALT AST Bilirubin BUN ConjugateSaline 30 76 0.2 40 440762 0.60 32 70 0.1 35 none 2 26 57 0.1 35 6 31 480.1 39 651900 0.2 32 115 0.2 39 GalNac₃-1 (3′) 0.6 33 61 0.1 35 2 30 500.1 37 6 34 52 0.1 36 663748 0.2 28 56 0.2 36 GalNac₄-11 (3′) 0.6 34 600.1 35 2 44 62 0.1 36 6 38 71 0.1 33

Example 59: Effects of GalNAc₃-1 Conjugated ASOs Targeting FXI In Vivo

The oligonucleotides listed below were tested in a multiple dose studyfor antisense inhibition of FXI in mice. ISIS 404071 was included as anunconjugated standard. Each of the conjugate groups was attached at the3′ terminus of the respective oligonucleotide by a phosphodiester linked2′-deoxyadenosine nucleoside cleavable moiety.

TABLE 49 Modified ASOs targeting FXI SEQ ID ASO Sequence (5′ to 3′)Linkages No. ISIS T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds) PS 146 404071 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(es)G_(es)A_(es)G_(es)G_(e) ISIST_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)^(m)C_(ds) PS 147 656172 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(es)G_(es)A_(es)G_(es)G_(eo) A _(do′)-GalNAc ₃-1 _(a) ISIST_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)^(m)C_(ds) PO/PS 147 656173 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(eo) A _(do′)-GalNAc ₃-1 _(a)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1 a was shown previously in Example 9.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously twice a week for 3 weeks at the dosage shownbelow with ISIS 404071, 656172, 656173 or with PBS treated control. Eachtreatment group consisted of 4 animals. The mice were sacrificed 72hours following the final administration to determine the liver FXI mRNAlevels using real-time PCR and RIBOGREEN® RNA quantification reagent(Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols.Plasma FXI protein levels were also measured using ELISA. FXI mRNAlevels were determined relative to total RNA (using RIBOGREEN®), priorto normalization to PBS-treated control. The results below are presentedas the average percent of FXI mRNA levels for each treatment group. Thedata was normalized to PBS-treated control and is denoted as “% PBS”.The ED₅₀s were measured using similar methods as described previouslyand are presented below.

TABLE 50 Factor XI mRNA (% Saline) Dose ASO mg/kg % Control ConjugateLinkages Saline 100 none ISIS 3 92 none PS 404071 10 40 30 15 ISIS 0.774 GalNAc₃-1 PS 656172 2 33 6 9 ISIS 0.7 49 GalNAc₃-1 PO/PS 656173 2 226 1

As illustrated in Table 50, treatment with antisense oligonucleotideslowered FXI mRNA levels in a dose-dependent manner. The oligonucleotidescomprising a 3′-GalNAc₃-1 conjugate group showed substantial improvementin potency compared to the unconjugated antisense oligonucleotide (ISIS404071). Between the two conjugated oligonucleotides an improvement inpotency was further provided by substituting some of the PS linkageswith PO (ISIS 656173).

As illustrated in Table 50a, treatment with antisense oligonucleotideslowered FXI protein levels in a dose-dependent manner. Theoligonucleotides comprising a 3′-GalNAc₃-1 conjugate group showedsubstantial improvement in potency compared to the unconjugatedantisense oligonucleotide (ISIS 404071). Between the two conjugatedoligonucleotides an improvement in potency was further provided bysubstituting some of the PS linkages with PO (ISIS 656173).

TABLE 50a Factor XI protein (% Saline) Dose Protein (% ASO mg/kgControl) Conjugate Linkages Saline 100 none ISIS 3 127 none PS 404071 1032 30 3 ISIS 0.7 70 GalNAc₃-1 PS 656172 2 23 6 1 ISIS 0.7 45 GalNAc₃-1PO/PS 656173 2 6 6 0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin, total albumin,CRE and BUN were also evaluated. The change in body weights wasevaluated with no significant change from the saline group. ALTs, ASTs,total bilirubin and BUN values are shown in the table below.

TABLE 51 Total Total ISIS No. Dosage mg/kg ALT AST Albumin Bilirubin CREBUN Conjugate Saline 71.8 84.0 3.1 0.2 0.2 22.9 404071 3 152.8 176.0 3.10.3 0.2 23.0 none 10 73.3 121.5 3.0 0.2 0.2 21.4 30 82.5 92.3 3.0 0.20.2 23.0 656172 0.7 62.5 111.5 3.1 0.2 0.2 23.8 GalNac₃-1 (3′) 2 33.051.8 2.9 0.2 0.2 22.0 6 65.0 71.5 3.2 0.2 0.2 23.9 656173 0.7 54.8 90.53.0 0.2 0.2 24.9 GalNac₃-1 (3′) 2 85.8 71.5 3.2 0.2 0.2 21.0 6 114.0101.8 3.3 0.2 0.2 22.7

Example 60: Effects of Conjugated ASOs Targeting SRB-1 In Vitro

The oligonucleotides listed below were tested in a multiple dose studyfor antisense inhibition of SRB-1 in primary mouse hepatocytes. ISIS353382 was included as an unconjugated standard. Each of the conjugategroups were attached at the 3′ or 5′ terminus of the respectiveoligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleosidecleavable moiety.

TABLE 52 Modified ASO targeting SRB-1 SEQ ASO Sequence (5′ to 3′) MotifConjugate ID No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5none 143 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃-1 144^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc₃-1 _(a) ISIS 655862 G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃-1 144 ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(eo)A _(do′)-GalNAc ₃-1 _(a) ISIS 661161 GalNAc ₃-3 _(a-o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃-3 145T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 665001 GalNAc ₃-8 _(a-o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc₃-8 145 T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 664078 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃-9 144^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc₃-9 _(a) ISIS 666961 GalNAc ₃-6 _(a)-_(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃-6 145T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 664507 GalNAc ₃-2 _(a)-_(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5GalNAc ₃-2 145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃-10 _(a)-_(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5GalNAc ₃-10 145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666224GalNAc ₃-5 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃-5 145^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666981 GalNAc ₃-7 _(a)-_(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃-7145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. Thestructure of GalNAc₃-3a was shown previously in Example 39. Thestructure of GalNAc₃-8a was shown previously in Example 47. Thestructure of GalNAc₃-9a was shown previously in Example 52. Thestructure of GalNAc₃-6a was shown previously in Example 51. Thestructure of GalNAc₃-2a was shown previously in Example 37. Thestructure of GalNAc₃-10a was shown previously in Example 46. Thestructure of GalNAc₃-5a was shown previously in Example 49. Thestructure of GalNAc₃-7a was shown previously in Example 48.

Treatment

The oligonucleotides listed above were tested in vitro in primary mousehepatocyte cells plated at a density of 25,000 cells per well andtreated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 or 20 nM modifiedoligonucleotide. After a treatment period of approximately 16 hours, RNAwas isolated from the cells and mRNA levels were measured byquantitative real-time PCR and the SRB-1 mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®.

The IC₅₀ was calculated using standard methods and the results arepresented in Table 53. The results show that, under free uptakeconditions in which no reagents or electroporation techniques are usedto artificially promote entry of the oligonucleotides into cells, theoligonucleotides comprising a GalNAc conjugate were significantly morepotent in hepatocytes than the parent oligonucleotide (ISIS 353382) thatdoes not comprise a GalNAc conjugate.

TABLE 53 Internucleoside SEQ ID ASO IC₅₀ (nM) linkages Conjugate No.ISIS 353382 190^(a)  PS none 143 ISIS 655861 11^(a) PS GalNAc₃-1 144ISIS 655862  3 PO/PS GalNAc₃-1 144 ISIS 661161 15^(a) PS GalNAc₃-3 145ISIS 665001 20 PS GalNAc₃-8 145 ISIS 664078 55 PS GalNAc₃-9 144 ISIS666961 22^(a) PS GalNAc₃-6 145 ISIS 664507 30 PS GalNAc₃-2 145 ISIS666881 30 PS  GalNAc₃-10 145 ISIS 666224 30^(a) PS GalNAc₃-5 145 ISIS666981 40 PS GalNAc₃-7 145 ^(a)Average of multiple runs.

Example 61: Preparation of Oligomeric Compound 175 Comprising GalNAc₃-12

Compound 169 is commercially available. Compound 172 was prepared byaddition of benzyl (perfluorophenyl) glutarate to compound 171. Thebenzyl (perfluorophenyl) glutarate was prepared by adding PFP-TFA andDIEA to 5-(benzyloxy)-5-oxopentanoic acid in DMF. Oligomeric compound175, comprising a GalNAc₃-12 conjugate group, was prepared from compound174 using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-12 (GalNAc₃-12_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In a certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-12(GalNAc₃-12_(a)-CM-) is shown below:

Example 62: Preparation of Oligomeric Compound 180 Comprising GalNAc₃-13

Compound 176 was prepared using the general procedure shown in Example2. Oligomeric compound 180, comprising a GalNAc₃-13 conjugate group, wasprepared from compound 177 using the general procedures illustrated inExample 49. The GalNAc₃ cluster portion of the conjugate groupGalNAc₃-13 (GalNAc₃-13_(a)) can be combined with any cleavable moiety toprovide a variety of conjugate groups. In a certain embodiments, thecleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure ofGalNAc₃-13 (GalNAc₃-13_(a)-CM-) is shown below:

Example 63: Preparation of Oligomeric Compound 188 Comprising GalNAc₃-14

Compounds 181 and 185 are commercially available. Oligomeric compound188, comprising a GalNAc₃-14 conjugate group, was prepared from compound187 using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-14 (GalNAc₃-14_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-14(GalNAc₃-14_(a)-CM-) is shown below:

Example 64: Preparation of Oligomeric Compound 197 Comprising GalNAc₃-15

Compound 189 is commercially available. Compound 195 was prepared usingthe general procedure shown in Example 31. Oligomeric compound 197,comprising a GalNAc₃-15 conjugate group, was prepared from compounds 194and 195 using standard oligonucleotide synthesis procedures. The GalNAc₃cluster portion of the conjugate group GalNAc₃-15 (GalNAc₃-15_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-15(GalNAc₃-15_(a)-CM-) is shown below:

Example 65: Dose-Dependent Study of Oligonucleotides Comprising a5′-Conjugate Group (Comparison of GalNAc₃-3, 12, 13, 14, and 15)Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 wasincluded as a standard. Each of the GalNAc₃ conjugate groups wasattached at the 5′ terminus of the respective oligonucleotide by aphosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety).

TABLE 54 Modified ASOs targeting SRB-1 SEQ ISIS ID No. Sequences (5′ to3′) Conjugate No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) none 143 661161GalNAc ₃-3 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-3 145 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)671144 GalNAc ₃-12 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-12 145 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)670061 GalNAc ₃-13 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-13 145 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)671261 GalNAc ₃-14 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-14 145 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)671262 GalNAc ₃-15 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-15 145 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-12a was shown previously in Example 61. Thestructure of GalNAc₃-13a was shown previously in Example 62. Thestructure of GalNAc₃-14a was shown previously in Example 63. Thestructure of GalNAc₃-15a was shown previously in Example 64.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once or twice at the dosage shown belowwith ISIS 353382, 661161, 671144, 670061, 671261, 671262, or withsaline. Mice that were dosed twice received the second dose three daysafter the first dose. Each treatment group consisted of 4 animals. Themice were sacrificed 72 hours following the final administration todetermine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN®RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)according to standard protocols. The results below are presented as theaverage percent of SRB-1 mRNA levels for each treatment group,normalized to the saline control.

As illustrated in Table 55, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. No significantdifferences in target knockdown were observed between animals thatreceived a single dose and animals that received two doses (see ISIS353382 dosages 30 and 2×15 mg/kg; and ISIS 661161 dosages 5 and 2×2.5mg/kg). The antisense oligonucleotides comprising the phosphodiesterlinked GalNAc₃-3, 12, 13, 14, and 15 conjugates showed substantialimprovement in potency compared to the unconjugated antisenseoligonucleotide (ISIS 335382).

TABLE 55 SRB-1 mRNA (% Saline) SRB-1 mRNA (% ISIS No. Dosage (mg/kg)Saline) ED₅₀ (mg/kg) Conjugate Saline n/a 100.0 n/a n/a 353382 3 85.022.4 none 10 69.2 30 34.2 2 × 15  36.0 661161 0.5 87.4 2.2 GalNAc₃-3 1.5 59.0 5 25.6 2 × 2.5 27.5 15 17.4 671144 0.5 101.2 3.4 GalNAc₃-12 1.576.1 5 32.0 15 17.6 670061 0.5 94.8 2.1 GalNAc₃-13 1.5 57.8 5 20.7 1513.3 671261 0.5 110.7 4.1 GalNAc₃-14 1.5 81.9 5 39.8 15 14.1 671262 0.5109.4 9.8 GalNAc₃-15 1.5 99.5 5 69.2 15 36.1

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The changes in body weights were evaluated with nosignificant differences from the saline group (data not shown). ALTs,ASTs, total bilirubin and BUN values are shown in Table 56 below.

TABLE 56 Total Dosage ALT Bilirubin BUN ISIS No. (mg/kg) (U/L) AST (U/L)(mg/dL) (mg/dL) Conjugate Saline n/a 28 60 0.1 39 n/a 353382 3 30 77 0.236 none 10 25 78 0.2 36 30 28 62 0.2 35 2 × 15 22 59 0.2 33 661161 0.539 72 0.2 34 GalNAc₃-3 1.5 26 50 0.2 33 5 41 80 0.2 32 2 × 2.5 24 72 0.228 15 32 69 0.2 36 671144 0.5 25 39 0.2 34 GalNAc₃-12 1.5 26 55 0.2 28 548 82 0.2 34 15 23 46 0.2 32 670061 0.5 27 53 0.2 33 GalNAc₃-13 1.5 2445 0.2 35 5 23 58 0.1 34 15 24 72 0.1 31 671261 0.5 69 99 0.1 33GalNAc₃-14 1.5 34 62 0.1 33 5 43 73 0.1 32 15 32 53 0.2 30 671262 0.5 2451 0.2 29 GalNAc₃-15 1.5 32 62 0.1 31 5 30 76 0.2 32 15 31 64 0.1 32

Example 66: Effect of Various Cleavable Moieties on Antisense InhibitionIn Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃Cluster

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Each of the GalNAc₃ conjugategroups was attached at the 5′ terminus of the respective oligonucleotideby a phosphodiester linked nucleoside (cleavable moiety (CM)).

TABLE 57 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 661161 GalNAc ₃-3 _(a)-_(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 145 G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 670699 GalNAc₃-3 _(a)-_(o′) T _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a T_(d) 148G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)670700 GalNAc ₃-3 _(a)-_(o′) A _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(e) 145G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)670701 GalNAc ₃-3 _(a)-_(o′) T _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a T_(e) 148G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)671165 GalNAc ₃-13 _(a)-_(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-13a A_(d)145 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-13a was shown previously in Example 62.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once at the dosage shown below with ISIS661161, 670699, 670700, 670701, 671165, or with saline. Each treatmentgroup consisted of 4 animals. The mice were sacrificed 72 hoursfollowing the final administration to determine the liver SRB-1 mRNAlevels using real-time PCR and RIBOGREEN® RNA quantification reagent(Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols.The results below are presented as the average percent of SRB-1 mRNAlevels for each treatment group, normalized to the saline control.

As illustrated in Table 58, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. The antisenseoligonucleotides comprising various cleavable moieties all showedsimilar potencies.

TABLE 58 SRB-1 mRNA (% Saline) SRB-1 mRNA GalNAc₃ ISIS No. Dosage(mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 661161 0.5 87.8GalNAc₃-3a A_(d) 1.5 61.3 5 33.8 15 14.0 670699 0.5 89.4 GalNAc₃-3aT_(d) 1.5 59.4 5 31.3 15 17.1 670700 0.5 79.0 GalNAc₃-3a A_(e) 1.5 63.35 32.8 15 17.9 670701 0.5 79.1 GalNAc₃-3a T_(e) 1.5 59.2 5 35.8 15 17.7671165 0.5 76.4  GalNAc₃-13a A_(d) 1.5 43.2 5 22.6 15 10.0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The changes in body weights were evaluated with nosignificant differences from the saline group (data not shown). ALTs,ASTs, total bilirubin and BUN values are shown in Table 56 below.

TABLE 59 Total Dosage ALT AST Bilirubin BUN GalNAc₃ ISIS No. (mg/kg)(U/L) (U/L) (mg/dL) (mg/dL) Cluster CM Saline n/a 24 64 0.2 31 n/a n/a661161 0.5 25 64 0.2 31 GalNAc₃-3a A_(d) 1.5 24 50 0.2 32 5 26 55 0.2 2815 27 52 0.2 31 670699 0.5 42 83 0.2 31 GalNAc₃-3a T_(d) 1.5 33 58 0.232 5 26 70 0.2 29 15 25 67 0.2 29 670700 0.5 40 74 0.2 27 GalNAc₃-3aA_(e) 1.5 23 62 0.2 27 5 24 49 0.2 29 15 25 87 0.1 25 670701 0.5 30 770.2 27 GalNAc₃-3a T_(e) 1.5 22 55 0.2 30 5 81 101 0.2 25 15 31 82 0.2 24671165 0.5 44 84 0.2 26 GalNAc₃-13a A_(d) 1.5 47 71 0.1 24 5 33 91 0.226 15 33 56 0.2 29

Example 67: Preparation of Oligomeric Compound 199 Comprising GalNAc₃-16

Oligomeric compound 199, comprising a GalNAc₃-16 conjugate group, isprepared using the general procedures illustrated in Examples 7 and 9.The GalNAc₃ cluster portion of the conjugate group GalNAc₃-16(GalNAc₃-16_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-16(GalNAc₃-16_(a)-CM-) is shown below:

Example 68: Preparation of Oligomeric Compound 200 Comprising GalNAc₃-17

Oligomeric compound 200, comprising a GalNAc₃-17 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-17(GalNAc₃-17_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-17(GalNAc₃-17_(a)-CM-) is shown below:

Example 69: Preparation of Oligomeric Compound 201 Comprising GalNAc₃-18

Oligomeric compound 201, comprising a GalNAc₃-18 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-18(GalNAc₃-18_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-18(GalNAc₃-18_(a)-CM-) is shown below:

Example 70: Preparation of Oligomeric Compound 204 Comprising GalNAc₃-19

Oligomeric compound 204, comprising a GalNAc₃-19 conjugate group, wasprepared from compound 64 using the general procedures illustrated inExample 52. The GalNAc₃ cluster portion of the conjugate groupGalNAc₃-19 (GalNAc₃-19_(a)) can be combined with any cleavable moiety toprovide a variety of conjugate groups. In certain embodiments, thecleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure ofGalNAc₃-19 (GalNAc₃-19_(a)-CM-) is shown below:

Example 71: Preparation of Oligomeric Compound 210 Comprising GalNAc₃-20

Compound 205 was prepared by adding PFP-TFA and DIEA to6-(2,2,2-trifluoroacetamido)hexanoic acid in acetonitrile, which wasprepared by adding triflic anhydride to 6-aminohexanoic acid. Thereaction mixture was heated to 80° C., then lowered to rt. Oligomericcompound 210, comprising a GalNAc₃-20 conjugate group, was prepared fromcompound 208 using the general procedures illustrated in Example 52. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-20(GalNAc₃-20_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-20(GalNAc₃-20_(a)-CM-) is shown below:

Example 72: Preparation of Oligomeric Compound 215 Comprising GalNAc₃-21

Compound 211 is commercially available. Oligomeric compound 215,comprising a GalNAc₃-21 conjugate group, was prepared from compound 213using the general procedures illustrated in Example 52. The GalNAc₃cluster portion of the conjugate group GalNAc₃-21 (GalNAc₃-21_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-21(GalNAc₃-21_(a)-CM-) is shown below:

Example 73: Preparation of Oligomeric Compound 221 Comprising GalNAc₃-22

Compound 220 was prepared from compound 219 using diisopropylammoniumtetrazolide. Oligomeric compound 221, comprising a GalNAc₃-21 conjugategroup, is prepared from compound 220 using the general procedureillustrated in Example 52. The GalNAc₃ cluster portion of the conjugategroup GalNAc₃-22 (GalNAc₃-22_(a)) can be combined with any cleavablemoiety to provide a variety of conjugate groups. In certain embodiments,the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure ofGalNAc₃-22 (GalNAc₃-22_(a)-CM-) is shown below:

Example 74: Effect of Various Cleavable Moieties on Antisense InhibitionIn Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃Conjugate

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Each of the GalNAc₃ conjugategroups was attached at the 5′ terminus of the respectiveoligonucleotide.

TABLE 60 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) n/a n/a 143 ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)661161 GalNAc ₃-3 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 145G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)666904 GalNAc ₃-3 _(a)-_(o′)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a PO 143G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)675441 GalNAc ₃-17 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-17a A_(d)145 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)675442 GalNAc ₃-18 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-18a A_(d)145 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)In all tables, capital letters indicate the nucleobase for eachnucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e”indicates a 2′-MOE modified nucleoside; “d” indicates aβ-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioateinternucleoside linkage (PS); “o” indicates a phosphodiesterinternucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—.Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-17a was shown previously in Example 68, and thestructure of GalNAc₃-18a was shown in Example 69.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once at the dosage shown below with anoligonucleotide listed in Table 60 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the SRB-1 mRNA levels using real-timePCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.Eugene, Oreg.) according to standard protocols. The results below arepresented as the average percent of SRB-1 mRNA levels for each treatmentgroup, normalized to the saline control.

As illustrated in Table 61, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. The antisenseoligonucleotides comprising a GalNAc conjugate showed similar potenciesand were significantly more potent than the parent oligonucleotidelacking a GalNAc conjugate.

TABLE 61 SRB-1 mRNA (% Saline) SRB-1 mRNA GalNAc₃ ISIS No. Dosage(mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 353382 3 79.38n/a n/a 10 68.67 30 40.70 661161 0.5 79.18 GalNAc₃-3a  A_(d) 1.5 75.96 530.53 15 12.52 666904 0.5 91.30 GalNAc₃-3a  PO 1.5 57.88 5 21.22 1516.49 675441 0.5 76.71 GalNAc₃-17a A_(d) 1.5 63.63 5 29.57 15 13.49675442 0.5 95.03 GalNAc₃-18a A_(d) 1.5 60.06 5 31.04 15 19.40

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group (data not shown). ALTs, ASTs,total bilirubin and BUN values are shown in Table 62 below.

TABLE 62 Total Dosage ALT AST Bilirubin BUN GalNAc₃ ISIS No. (mg/kg)(U/L) (U/L) (mg/dL) (mg/dL) Cluster CM Saline n/a 26 59 0.16 42 n/a n/a353382 3 23 58 0.18 39 n/a n/a 10 28 58 0.16 43 30 20 48 0.12 34 6611610.5 30 47 0.13 35 GalNAc₃-3a A_(d) 1.5 23 53 0.14 37 5 26 48 0.15 39 1532 57 0.15 42 666904 0.5 24 73 0.13 36 GalNAc₃-3a PO 1.5 21 48 0.12 32 519 49 0.14 33 15 20 52 0.15 26 675441 0.5 42 148 0.21 36 GalNAc₃-17aA_(d) 1.5 60 95 0.16 34 5 27 75 0.14 37 15 24 61 0.14 36 675442 0.5 2665 0.15 37 GalNAc₃-18a A_(d) 1.5 25 64 0.15 43 5 27 69 0.15 37 15 30 840.14 37

Example 75: Pharmacokinetic Analysis of Oligonucleotides Comprising a5′-Conjugate Group

The PK of the ASOs in Tables 54, 57 and 60 above was evaluated usingliver samples that were obtained following the treatment proceduresdescribed in Examples 65, 66, and 74. The liver samples were minced andextracted using standard protocols and analyzed by IP-HPLC-MS alongsidean internal standard. The combined tissue level (μg/g) of allmetabolites was measured by integrating the appropriate UV peaks, andthe tissue level of the full-length ASO missing the conjugate (“parent,”which is Isis No. 353382 in this case) was measured using theappropriate extracted ion chromatograms (EIC).

TABLE 63 PK Analysis in Liver Total Tissue Parent ASO ISIS Dosage Levelby Tissue Level GalNAc₃ No. (mg/kg) UV (μg/g) by EIC (μg/g) Cluster CM353382 3 8.9 8.6 n/a n/a 10 22.4 21.0 30 54.2 44.2 661161 5 32.4 20.7GalNAc₃-3a A_(d) 15 63.2 44.1 671144 5 20.5 19.2  GalNAc₃-12a A_(d) 1548.6 41.5 670061 5 31.6 28.0  GalNAc₃-13a A_(d) 15 67.6 55.5 671261 519.8 16.8  GalNAc₃-14a A_(d) 15 64.7 49.1 671262 5 18.5 7.4  GalNAc₃-15aA_(d) 15 52.3 24.2 670699 5 16.4 10.4 GalNAc₃-3a T_(d) 15 31.5 22.5670700 5 19.3 10.9 GalNAc₃-3a A_(e) 15 38.1 20.0 670701 5 21.8 8.8GalNAc₃-3a T_(e) 15 35.2 16.1 671165 5 27.1 26.5  GalNAc₃-13a A_(d) 1548.3 44.3 666904 5 30.8 24.0 GalNAc₃-3a PO 15 52.6 37.6 675441 5 25.419.0  GalNAc₃-17a A_(d) 15 54.2 42.1 675442 5 22.2 20.7  GalNAc₃-18aA_(d) 15 39.6 29.0

The results in Table 63 above show that there were greater liver tissuelevels of the oligonucleotides comprising a GalNAc₃ conjugate group thanof the parent oligonucleotide that does not comprise a GalNAc₃ conjugategroup (ISIS 353382) 72 hours following oligonucleotide administration,particularly when taking into consideration the differences in dosingbetween the oligonucleotides with and without a GalNAc₃ conjugate group.Furthermore, by 72 hours, 40-98% of each oligonucleotide comprising aGalNAc₃ conjugate group was metabolized to the parent compound,indicating that the GalNAc₃ conjugate groups were cleaved from theoligonucleotides.

Example 76: Preparation of Oligomeric Compound 230 Comprising GalNAc₃-23

Compound 222 is commercially available. 44.48 ml (0.33 mol) of compound222 was treated with tosyl chloride (25.39 g, 0.13 mol) in pyridine (500mL) for 16 hours. The reaction was then evaporated to an oil, dissolvedin EtOAc and washed with water, sat. NaHCO₃, brine, and dried overNa₂SO₄. The ethyl acetate was concentrated to dryness and purified bycolumn chromatography, eluted with EtOAc/hexanes (1:1) followed by 10%methanol in CH₂Cl₂ to give compound 223 as a colorless oil. LCMS and NMRwere consistent with the structure. 10 g (32.86 mmol) of1-Tosyltriethylene glycol (compound 223) was treated with sodium azide(10.68 g, 164.28 mmol) in DMSO (100 mL) at room temperature for 17hours. The reaction mixture was then poured onto water, and extractedwith EtOAc. The organic layer was washed with water three times anddried over Na₂SO₄. The organic layer was concentrated to dryness to give5.3 g of compound 224 (92%). LCMS and NMR were consistent with thestructure. 1-Azidotriethylene glycol (compound 224, 5.53 g, 23.69 mmol)and compound 4 (6 g, 18.22 mmol) were treated with 4A molecular sieves(5 g), and TMSOTf (1.65 ml, 9.11 mmol) in dichloromethane (100 mL) underan inert atmosphere. After 14 hours, the reaction was filtered to removethe sieves, and the organic layer was washed with sat. NaHCO₃, water,brine, and dried over Na₂SO₄. The organic layer was concentrated todryness and purified by column chromatography, eluted with a gradient of2 to 4% methanol in dichloromethane to give compound 225. LCMS and NMRwere consistent with the structure. Compound 225 (11.9 g, 23.59 mmol)was hydrogenated in EtOAc/Methanol (4:1, 250 mL) over Pearlman'scatalyst. After 8 hours, the catalyst was removed by filtration and thesolvents removed to dryness to give compound 226. LCMS and NMR wereconsistent with the structure.

In order to generate compound 227, a solution ofnitromethanetrispropionic acid (4.17 g, 15.04 mmol) and Hunig's base(10.3 ml, 60.17 mmol) in DMF (100 mL) were treated dropwise withpentaflourotrifluoro acetate (9.05 ml, 52.65 mmol). After 30 minutes,the reaction was poured onto ice water and extracted with EtOAc. Theorganic layer was washed with water, brine, and dried over Na₂SO₄. Theorganic layer was concentrated to dryness and then recrystallized fromheptane to give compound 227 as a white solid. LCMS and NMR wereconsistent with the structure. Compound 227 (1.5 g, 1.93 mmol) andcompound 226 (3.7 g, 7.74 mmol) were stirred at room temperature inacetonitrile (15 mL) for 2 hours. The reaction was then evaporated todryness and purified by column chromatography, eluting with a gradientof 2 to 10% methanol in dichloromethane to give compound 228. LCMS andNMR were consistent with the structure. Compound 228 (1.7 g, 1.02 mmol)was treated with Raney Nickel (about 2 g wet) in ethanol (100 mL) in anatmosphere of hydrogen. After 12 hours, the catalyst was removed byfiltration and the organic layer was evaporated to a solid that was useddirectly in the next step. LCMS and NMR were consistent with thestructure. This solid (0.87 g, 0.53 mmol) was treated withbenzylglutaric acid (0.18 g, 0.8 mmol), HBTU (0.3 g, 0.8 mmol) and DIEA(273.7 μl, 1.6 mmol) in DMF (5 mL). After 16 hours, the DMF was removedunder reduced pressure at 65° C. to an oil, and the oil was dissolved indichloromethane. The organic layer was washed with sat. NaHCO₃, brine,and dried over Na₂SO₄. After evaporation of the organic layer, thecompound was purified by column chromatography and eluted with agradient of 2 to 20% methanol in dichloromethane to give the coupledproduct. LCMS and NMR were consistent with the structure. The benzylester was deprotected with Pearlman's catalyst under a hydrogenatmosphere for 1 hour. The catalyst was them removed by filtration andthe solvents removed to dryness to give the acid. LCMS and NMR wereconsistent with the structure. The acid (486 mg, 0.27 mmol) wasdissolved in dry DMF (3 mL). Pyridine (53.61 μl, 0.66 mmol) was addedand the reaction was purged with argon. Pentaflourotriflouro acetate(46.39 μl, 0.4 mmol) was slowly added to the reaction mixture. The colorof the reaction changed from pale yellow to burgundy, and gave off alight smoke which was blown away with a stream of argon. The reactionwas allowed to stir at room temperature for one hour (completion ofreaction was confirmed by LCMS). The solvent was removed under reducedpressure (rotovap) at 70° C. The residue was diluted with DCM and washedwith 1N NaHSO₄, brine, saturated sodium bicarbonate and brine again. Theorganics were dried over Na₂SO₄, filtered, and were concentrated todryness to give 225 mg of compound 229 as a brittle yellow foam. LCMSand NMR were consistent with the structure.

Oligomeric compound 230, comprising a GalNAc₃-23 conjugate group, wasprepared from compound 229 using the general procedure illustrated inExample 46. The GalNAc₃ cluster portion of the GalNAc₃-23 conjugategroup (GalNAc₃-23_(a)) can be combined with any cleavable moiety toprovide a variety of conjugate groups. The structure of GalNAc₃-23(GalNAc₃-23_(a)-CM) is shown below:

Example 77: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising a GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice.

TABLE 64 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 661161 GalNAc ₃-3 _(a)-_(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 145 G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666904 GalNAc₃-3 _(a)-_(o′)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a PO 143 G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 673502 GalNAc₃-10 _(a)-_(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-10a A_(d)145 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)677844 GalNAc ₃-9 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-9a A_(d) 145G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)677843 GalNAc ₃-23 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-23a A_(d)145 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)GalNAc₃-1a A_(d) 144 ^(m)C_(es)T_(es)T_(eo)A_(do′)-GalNAc₃-1_(a) 677841G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)GalNAc₃-19a A_(d) 144 ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃-19 _(a)677842 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)GalNAc₃-20a A_(d) 144 ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃-20 _(a)

The structure of GalNAc₃-1 a was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-9a was shown in Example52, GalNAc₃-10a was shown in Example 46, GalNAc₃-19_(a) was shown inExample 70, GalNAc₃-20_(a) was shown in Example 71, and GalNAc₃-23_(a)was shown in Example 76.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were each injected subcutaneously once at a dosage shown below with anoligonucleotide listed in Table 64 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the SRB-1 mRNA levels using real-timePCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.Eugene, Oreg.) according to standard protocols. The results below arepresented as the average percent of SRB-1 mRNA levels for each treatmentgroup, normalized to the saline control.

As illustrated in Table 65, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 65 SRB-1 mRNA (% Saline) SRB-1 mRNA GalNAc₃ ISIS No. Dosage(mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 661161 0.5 89.18GalNAc₃-3a A_(d) 1.5 77.02 5 29.10 15 12.64 666904 0.5 93.11 GalNAc₃-3aPO 1.5 55.85 5 21.29 15 13.43 673502 0.5 77.75  GalNAc₃-10a A_(d) 1.541.05 5 19.27 15 14.41 677844 0.5 87.65 GalNAc₃-9a A_(d) 1.5 93.04 540.77 15 16.95 677843 0.5 102.28  GalNAc₃-23a A_(d) 1.5 70.51 5 30.68 1513.26 655861 0.5 79.72 GalNAc₃-1a A_(d) 1.5 55.48 5 26.99 15 17.58677841 0.5 67.43  GalNAc₃-19a A_(d) 1.5 45.13 5 27.02 15 12.41 6778420.5 64.13  GalNAc₃-20a A_(d) 1.5 53.56 5 20.47 15 10.23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were also measured using standardprotocols. Total bilirubin and BUN were also evaluated. Changes in bodyweights were evaluated, with no significant change from the saline group(data not shown). ALTs, ASTs, total bilirubin and BUN values are shownin Table 66 below.

TABLE 66 Total Dosage ALT AST Bilirubin BUN GalNAc₃ ISIS No. (mg/kg)(U/L) (U/L) (mg/dL) (mg/dL) Cluster CM Saline n/a 21 45 0.13 34 n/a n/a661161 0.5 28 51 0.14 39 GalNAc₃-3a A_(d) 1.5 23 42 0.13 39 5 22 59 0.1337 15 21 56 0.15 35 666904 0.5 24 56 0.14 37 GalNAc₃-3a PO 1.5 26 680.15 35 5 23 77 0.14 34 15 24 60 0.13 35 673502 0.5 24 59 0.16 34GalNAc₃-10a A_(d) 1.5 20 46 0.17 32 5 24 45 0.12 31 15 24 47 0.13 34677844 0.5 25 61 0.14 37 GalNAc₃-9a A_(d) 1.5 23 64 0.17 33 5 25 58 0.1335 15 22 65 0.14 34 677843 0.5 53 53 0.13 35 GalNAc₃-23a A_(d) 1.5 25 540.13 34 5 21 60 0.15 34 15 22 43 0.12 38 655861 0.5 21 48 0.15 33GalNAc₃-1a A_(d) 1.5 28 54 0.12 35 5 22 60 0.13 36 15 21 55 0.17 30677841 0.5 32 54 0.13 34 GalNAc₃-19a A_(d) 1.5 24 56 0.14 34 5 23 920.18 31 15 24 58 0.15 31 677842 0.5 23 61 0.15 35 GalNAc₃-20a A_(d) 1.524 57 0.14 34 5 41 62 0.15 35 15 24 37 0.14 32

Example 78: Antisense Inhibition In Vivo by Oligonucleotides TargetingAngiotensinogen Comprising a GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of Angiotensinogen (AGT) in normotensiveSprague Dawley rats.

TABLE 67 Modified ASOs targeting AGT ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 552668 ^(m)C_(es)A_(es)^(m)C_(es)T_(es)G_(es)A_(ds)T_(ds)T_(ds)T_(ds)T_(ds)T_(ds)G_(ds)^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(es)G_(es) n/a n/a 149G_(es)A_(es)T_(e) 669509 ^(m)C_(es)A_(es)^(m)C_(es)T_(es)G_(es)A_(ds)T_(ds)T_(ds)T_(ds)T_(ds)T_(ds)G_(ds)^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(es)G_(es) GalNAc₃-1_(a) A_(d) 150G_(es)A_(es)T_(eo) A _(do′)-GalNAc ₃-1 _(a)The structure of GalNAc₃-1 a was shown previously in Example 9.Treatment

Six week old, male Sprague Dawley rats were each injected subcutaneouslyonce per week at a dosage shown below, for a total of three doses, withan oligonucleotide listed in Table 67 or with PBS. Each treatment groupconsisted of 4 animals. The rats were sacrificed 72 hours following thefinal dose. AGT liver mRNA levels were measured using real-time PCR andRIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene,Oreg.) according to standard protocols. AGT plasma protein levels weremeasured using the Total Angiotensinogen ELISA (Catalog # JP27412, IBLInternational, Toronto, ON) with plasma diluted 1:20,000. The resultsbelow are presented as the average percent of AGT mRNA levels in liveror AGT protein levels in plasma for each treatment group, normalized tothe PBS control.

As illustrated in Table 68, treatment with antisense oligonucleotideslowered AGT liver mRNA and plasma protein levels in a dose-dependentmanner, and the oligonucleotide comprising a GalNAc conjugate wassignificantly more potent than the parent oligonucleotide lacking aGalNAc conjugate.

TABLE 68 AGT liver mRNA and plasma protein levels AGT liver AGT plasmaISIS Dosage mRNA protein GalNAc₃ No. (mg/kg) (% PBS) (% PBS) Cluster CMPBS n/a 100 100 n/a n/a 552668 3 95 122 n/a n/a 10 85 97 30 46 79 90 811 669509 0.3 95 70 GalNAc₃-1a A_(d) 1 95 129 3 62 97 10 9 23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in plasma and body weights were also measured attime of sacrifice using standard protocols. The results are shown inTable 69 below.

TABLE 69 Liver transaminase levels and rat body weights Body Dosage ALTAST Weight (% GalNAc₃ ISIS No. (mg/kg) (U/L) (U/L) of baseline) ClusterCM PBS n/a 51 81 186 n/a n/a 552668 3 54 93 183 n/a n/a 10 51 93 194 3059 99 182 90 56 78 170 669509 0.3 53 90 190 GalNAc₃-1a A_(d) 1 51 93 1923 48 85 189 10 56 95 189

Example 79: Duration of Action In Vivo of Oligonucleotides TargetingAPOC-III Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 70 below were tested in a singledose study for duration of action in mice.

TABLE 70 Modified ASOs targeting APOC-III ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 304801 A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es) n/a n/a 135 T_(es)A_(es)T_(e) 647535 A_(es)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) GalNAc₃-1a A_(d) 136T_(es)A_(es)T_(eo) A _(do′)-GalNAc ₃-1 _(a) 663083 GalNAc ₃-3 _(a)-_(o′)A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-3a A_(d) 151^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674449GalNAc ₃-7 _(a)-_(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-7a A_(d) 151^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674450GalNAc ₃-10 _(a)-_(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-10a A_(d) 151^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674451GalNAc ₃-13 _(a)-_(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-13a A_(d) 151^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e)The structure of GalNAc₃-1 a was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a)was shown in Example 62.Treatment

Six to eight week old transgenic mice that express human APOC-III wereeach injected subcutaneously once with an oligonucleotide listed inTable 70 or with PBS. Each treatment group consisted of 3 animals. Bloodwas drawn before dosing to determine baseline and at 72 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the dose. Plasmatriglyceride and APOC-III protein levels were measured as described inExample 20. The results below are presented as the average percent ofplasma triglyceride and APOC-III levels for each treatment group,normalized to baseline levels, showing that the oligonucleotidescomprising a GalNAc conjugate group exhibited a longer duration ofaction than the parent oligonucleotide without a conjugate group (ISIS304801) even though the dosage of the parent was three times the dosageof the oligonucleotides comprising a GalNAc conjugate group.

TABLE 71 Plasma triglyceride and APOC-III protein levels in transgenicmice Time point Triglyc- APOC-III (days erides protein ISIS Dosage post-(% (% GalNAc₃ No. (mg/kg) dose) baseline) baseline) Cluster CM PBS n/a 397 102 n/a n/a 7 101 98 14 108 98 21 107 107 28 94 91 35 88 90 42 91 105304801 30 3 40 34 n/a n/a 7 41 37 14 50 57 21 50 50 28 57 73 35 68 70 4275 93 647535 10 3 36 37 GalNAc₃-1a A_(d) 7 39 47 14 40 45 21 41 41 28 4262 35 69 69 42 85 102 663083 10 3 24 18 GalNAc₃-3a A_(d) 7 28 23 14 2527 21 28 28 28 37 44 35 55 57 42 60 78 674449 10 3 29 26 GalNAc₃-7aA_(d) 7 32 31 14 38 41 21 44 44 28 53 63 35 69 77 42 78 99 674450 10 333 30 GalNAc₃-10a A_(d) 7 35 34 14 31 34 21 44 44 28 56 61 35 68 70 4283 95 674451 10 3 35 33 GalNAc₃-13a A_(d) 7 24 32 14 40 34 21 48 48 2854 67 35 65 75 42 74 97

Example 80: Antisense Inhibition In Vivo by Oligonucleotides TargetingAlpha-1 Antitrypsin (A1AT) Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 72 below were tested in a study fordose-dependent inhibition of A1AT in mice.

TABLE 72 Modified ASOs targeting A1AT ISIS GalNAc₃ SEQ ID No. Sequences(5′ to 3′) Cluster CM No. 476366 A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(ds)G_(ds)A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es) n/a n/a 152G_(es)G_(es)A_(e) 656326 A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(ds)G_(ds)A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es) GalNAc₃-1aA_(d) 153 G_(es)G_(es)A_(eo) A _(do′)-GalNAc ₃-1 _(a) 678381 GalNAc ₃-3_(a)-_(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds)GalNAc₃-3a A_(d) 154 A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e)678382 GalNAc ₃-7 _(a)-_(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds)GalNAc₃-7a A_(d) 154 A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e)678383 GalNAc ₃-10 _(a)-_(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) GalNAc₃-10aA_(d) 154 A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e) 678384GalNAc ₃-13 _(a)-_(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) GalNAc₃-13aA_(d) 154 A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a)was shown in Example 62.Treatment

Six week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were each injected subcutaneously once per week at a dosage shown below,for a total of three doses, with an oligonucleotide listed in Table 72or with PBS. Each treatment group consisted of 4 animals. The mice weresacrificed 72 hours following the final administration. A1AT liver mRNAlevels were determined using real-time PCR and RIBOGREEN® RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.) accordingto standard protocols. A1AT plasma protein levels were determined usingthe Mouse Alpha 1-Antitrypsin ELISA (catalog #41-A1AMS-E01, Alpco,Salem, N.H.). The results below are presented as the average percent ofA1AT liver mRNA and plasma protein levels for each treatment group,normalized to the PBS control.

As illustrated in Table 73, treatment with antisense oligonucleotideslowered A1AT liver mRNA and A1AT plasma protein levels in adose-dependent manner. The oligonucleotides comprising a GalNAcconjugate were significantly more potent than the parent (ISIS 476366).

TABLE 73 A1AT liver mRNA and plasma protein levels ISIS Dosage A1ATliver A1AT plasma GalNAc₃ No. (mg/kg) mRNA (% PBS) protein (% PBS)Cluster CM PBS n/a 100 100 n/a n/a 476366 5 86 78 n/a n/a 15 73 61 45 3038 656326 0.6 99 90 GalNAc₃-1a A_(d) 2 61 70 6 15 30 18 6 10 678381 0.6105 90 GalNAc₃-3a A_(d) 2 53 60 6 16 20 18 7 13 678382 0.6 90 79GalNAc₃-7a A_(d) 2 49 57 6 21 27 18 8 11 678383 0.6 94 84  GalNAc₃-10aA_(d) 2 44 53 6 13 24 18 6 10 678384 0.6 106 91  GalNAc₃-13a A_(d) 2 6559 6 26 31 18 11 15

Liver transaminase and BUN levels in plasma were measured at time ofsacrifice using standard protocols. Body weights and organ weights werealso measured. The results are shown in Table 74 below. Body weight isshown as % relative to baseline. Organ weights are shown as % of bodyweight relative to the PBS control group.

TABLE 74 Body Liver Kidney Spleen ISIS Dosage ALT AST BUN weight (%weight (Rel weight (Rel weight (Rel No. (mg/kg) (U/L) (U/L) (mg/dL)baseline) % BW) % BW) % BW) PBS n/a 25 51 37 119 100 100 100 476366 5 3468 35 116 91 98 106 15 37 74 30 122 92 101 128 45 30 47 31 118 99 108123 656326 0.6 29 57 40 123 100 103 119 2 36 75 39 114 98 111 106 6 3267 39 125 99 97 122 18 46 77 36 116 102 109 101 678381 0.6 26 57 32 11793 109 110 2 26 52 33 121 96 106 125 6 40 78 32 124 92 106 126 18 31 5428 118 94 103 120 678382 0.6 26 42 35 114 100 103 103 2 25 50 31 117 91104 117 6 30 79 29 117 89 102 107 18 65 112 31 120 89 104 113 678383 0.630 67 38 121 91 100 123 2 33 53 33 118 98 102 121 6 32 63 32 117 97 105105 18 36 68 31 118 99 103 108 678384 0.6 36 63 31 118 98 103 98 2 32 6132 119 93 102 114 6 34 69 34 122 100 100 96 18 28 54 30 117 98 101 104

Example 81: Duration of Action In Vivo of Oligonucleotides TargetingA1AT Comprising a GalNAc₃ Cluster

The oligonucleotides listed in Table 72 were tested in a single dosestudy for duration of action in mice.

Treatment

Six week old, male C57BL/6 mice were each injected subcutaneously oncewith an oligonucleotide listed in Table 72 or with PBS. Each treatmentgroup consisted of 4 animals. Blood was drawn the day before dosing todetermine baseline and at 5, 12, 19, and 25 days following the dose.Plasma A1AT protein levels were measured via ELISA (see Example 80). Theresults below are presented as the average percent of plasma A1ATprotein levels for each treatment group, normalized to baseline levels.The results show that the oligonucleotides comprising a GalNAc conjugatewere more potent and had longer duration of action than the parentlacking a GalNAc conjugate (ISIS 476366). Furthermore, theoligonucleotides comprising a 5′-GalNAc conjugate (ISIS 678381, 678382,678383, and 678384) were generally even more potent with even longerduration of action than the oligonucleotide comprising a 3′-GalNAcconjugate (ISIS 656326).

TABLE 75 Plasma A1AT protein levels in mice Time point ISIS Dosage (dayspost- A1AT (% GalNAc₃ No. (mg/kg) dose) baseline) Cluster CM PBS n/a 593 n/a n/a 12 93 19 90 25 97 476366 100 5 38 n/a n/a 12 46 19 62 25 77656326 18 5 33 GalNAc₃-1a A_(d) 12 36 19 51 25 72 678381 18 5 21GalNAc₃-3a A_(d) 12 21 19 35 25 48 678382 18 5 21 GalNAc₃-7a A_(d) 12 2119 39 25 60 678383 18 5 24  GalNAc₃-10a A_(d) 12 21 19 45 25 73 67838418 5 29  GalNAc₃-13a A_(d) 12 34 19 57 25 76

Example 82: Antisense Inhibition In Vitro by Oligonucleotides TargetingSRB-1 Comprising a GalNAc₃ Conjugate

Primary mouse liver hepatocytes were seeded in 96 well plates at 15,000cells/well 2 hours prior to treatment. The oligonucleotides listed inTable 76 were added at 2, 10, 50, or 250 nM in Williams E medium andcells were incubated overnight at 37° C. in 5% CO₂. Cells were lysed 16hours following oligonucleotide addition, and total RNA was purifiedusing RNease 3000 BioRobot (Qiagen). SRB-1 mRNA levels were determinedusing real-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. IC₅₀ valueswere determined using Prism 4 software (GraphPad). The results show thatoligonucleotides comprising a variety of different GalNAc conjugategroups and a variety of different cleavable moieties are significantlymore potent in an in vitro free uptake experiment than the parentoligonucleotides lacking a GalNAc conjugate group (ISIS 353382 and666841).

TABLE 76 Inhibition of SRB-1 expression in vitro ISIS GalNAc IC₅₀ SEQNo. Sequence (5′ to 3′) Linkages cluster CM (nM) ID No. 353382 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS n/a n/a 250 143^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 40 144^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc₃-1 _(a) 1_(a) 661161 GalNAc ₃-3 _(a)-_(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 40145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 3_(a) 661162 GalNAc ₃-3 _(a)-_(o′) A _(do)G_(es)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d)8 145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo)^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 664078 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 20 144^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc₃-9 _(a) 9_(a) 665001 GalNAc ₃-8 _(a)-_(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 70145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 8_(a) 666224 GalNAc ₃-5 _(a)-_(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 80145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 5_(a) 666841 G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PO/PSn/a n/a >250 143 ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)666881 GalNAc ₃-10 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 145^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 10_(a) 666904 GalNAc₃-3_(a)-_(o′)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) PSGalNAc₃- PO 9 143 A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 666924GalNAc₃-3_(a)-_(o′)T_(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- T_(d) 15 148^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 3_(a) 666961 GalNAc ₃-6 _(a)-_(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d)150 145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 6_(a) 666981 GalNAc ₃-7 _(a)-_(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 20 145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 7_(a) 670061GalNAc ₃-13 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 145^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 13_(a) 670699 GalNAc₃-3_(a)-_(o′)T_(do)G_(es)^(m)C_(es)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- T_(d)15 148 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 670700GalNAc₃-3_(a)-_(o′)A_(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(e) 30 145^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(eo)^(m)C_(es)T_(es)T 3_(a) 670701 GalNAc₃-3_(a)-_(o′)T_(eo)G_(es)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- T_(e)25 148 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo)^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 671144 GalNAc ₃-12 _(a)-_(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 40 145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 12_(a) 671165GalNAc ₃-13 _(a)-_(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 8 145^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(eo)^(m)C_(es)T_(es)T 13_(a) 671261 GalNAc ₃-14 _(a)-_(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃-A_(d) >250 145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 14_(a) 671262 GalNAc ₃-15 _(a)-_(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) >250 145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 15_(a) 673501GalNAc ₃-7 _(a)-_(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 30 145^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)^(m)C_(es)T_(es)T_(e) 7_(a) 673502 GalNAc ₃-10 _(a)-_(o′) A _(do)G_(es)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d)8 145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo)^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 10_(a) 675441 GalNAc ₃-17 _(a)-_(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 30 145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 17_(a) 675442GalNAc ₃-18 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 20 145^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 18_(a) 677841 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PSGalNAc₃- A_(d) 40 144 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃-19 _(a) 19_(a) 677842 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 30 144^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc₃-20 _(a) 20_(a) 677843 GalNAc ₃-23 _(a)-_(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 40145 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 23_(a)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-5_(a) was shown inExample 49, GalNAc₃-6_(a) was shown in Example 51, GalNAc₃-7_(a) wasshown in Example 48, GalNAc₃-8_(a) was shown in Example 47,GalNAc₃-9_(a) was shown in Example 52, GalNAc₃-10_(a) was shown inExample 46, GalNAc₃-12_(a) was shown in Example 61, GalNAc₃-13_(a) wasshown in Example 62, GalNAc₃-14_(a) was shown in Example 63,GalNAc₃-15_(a) was shown in Example 64, GalNAc₃-17_(a) was shown inExample 68, GalNAc₃-18_(a) was shown in Example 69, GalNAc₃-19_(a) wasshown in Example 70, GalNAc₃-20_(a) was shown in Example 71, andGalNAc₃-23_(a) was shown in Example 76.

Example 83: Antisense Inhibition In Vivo by Oligonucleotides TargetingFactor XI Comprising a GalNAc₃ Cluster

The oligonucleotides listed in Table 77 below were tested in a study fordose-dependent inhibition of Factor XI in mice.

TABLE 77 Modified oligonucleotides targeting Factor XI ISIS GalNAc SEQNo. Sequence (5′ to 3′) cluster CM ID No. 404071T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(es)G_(es) n/a n/a 146A_(es)G_(es)G_(e) 656173 T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo) GalNAc₃-1_(a) A_(d) 147 A_(es)G_(es)G_(eo) A_(do′)-GalNAc ₃-1 _(a) 663086 GalNAc ₃-3 _(a)-_(o′) A_(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-3_(a) A_(d) 155 T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678347 GalNAc ₃-7 _(a)-_(o′) A_(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-7_(a) A_(d) 155 T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678348 GalNAc ₃-10 _(a)-_(o′) A_(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-10_(a) A_(d) 155 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678349 GalNAc ₃-13 _(a)-_(o′) A_(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-13_(a) A_(d) 155 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e)The structure of GalNAc₃-1 a was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a)was shown in Example 62.Treatment

Six to eight week old mice were each injected subcutaneously once perweek at a dosage shown below, for a total of three doses, with anoligonucleotide listed below or with PBS. Each treatment group consistedof 4 animals. The mice were sacrificed 72 hours following the finaldose. Factor XI liver mRNA levels were measured using real-time PCR andnormalized to cyclophilin according to standard protocols. Livertransaminases, BUN, and bilirubin were also measured. The results beloware presented as the average percent for each treatment group,normalized to the PBS control.

As illustrated in Table 78, treatment with antisense oligonucleotideslowered Factor XI liver mRNA in a dose-dependent manner. The resultsshow that the oligonucleotides comprising a GalNAc conjugate were morepotent than the parent lacking a GalNAc conjugate (ISIS 404071).Furthermore, the oligonucleotides comprising a 5′-GalNAc conjugate (ISIS663086, 678347, 678348, and 678349) were even more potent than theoligonucleotide comprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 78 Factor XI liver mRNA, liver transaminase, BUN, and bilirubinlevels ISIS Dosage Factor XI ALT AST BUN Bilirubin GalNAc₃ SEQ No.(mg/kg) mRNA (% PBS) (U/L) (U/L) (mg/dL) (mg/dL) Cluster ID No. PBS n/a100 63 70 21 0.18 n/a n/a 404071 3 65 41 58 21 0.15 n/a 146 10 33 49 5323 0.15 30 17 43 57 22 0.14 656173 0.7 43 90 89 21 0.16 GalNAc₃-1a 147 29 36 58 26 0.17 6 3 50 63 25 0.15 663086 0.7 33 91 169 25 0.16GalNAc₃-3a 155 2 7 38 55 21 0.16 6 1 34 40 23 0.14 678347 0.7 35 28 4920 0.14 GalNAc₃-7a 155 2 10 180 149 21 0.18 6 1 44 76 19 0.15 678348 0.739 43 54 21 0.16 GalNAc₃-10a 155 2 5 38 55 22 0.17 6 2 25 38 20 0.14678349 0.7 34 39 46 20 0.16 GalNAc₃-13a 155 2 8 43 63 21 0.14 6 2 28 4120 0.14

Example 84: Duration of Action In Vivo of Oligonucleotides TargetingFactor XI Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 77 were tested in a single dosestudy for duration of action in mice.

Treatment

Six to eight week old mice were each injected subcutaneously once withan oligonucleotide listed in Table 77 or with PBS. Each treatment groupconsisted of 4 animals. Blood was drawn by tail bleeds the day beforedosing to determine baseline and at 3, 10, and 17 days following thedose. Plasma Factor XI protein levels were measured by ELISA usingFactor XI capture and biotinylated detection antibodies from R & DSystems, Minneapolis, Minn. (catalog # AF2460 and # BAF2460,respectively) and the OptEIA Reagent Set B (Catalog #550534, BDBiosciences, San Jose, Calif.). The results below are presented as theaverage percent of plasma Factor XI protein levels for each treatmentgroup, normalized to baseline levels. The results show that theoligonucleotides comprising a GalNAc conjugate were more potent withlonger duration of action than the parent lacking a GalNAc conjugate(ISIS 404071). Furthermore, the oligonucleotides comprising a 5′-GalNAcconjugate (ISIS 663086, 678347, 678348, and 678349) were even morepotent with an even longer duration of action than the oligonucleotidecomprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 79 Plasma Factor XI protein levels in mice Time point Factor XISEQ ISIS Dosage (days (% GalNAc₃ ID No. (mg/kg) post-dose) baseline)Cluster CM No. PBS n/a 3 123 n/a n/a n/a 10 56 17 100 404071 30 3 11 n/an/a 146 10 47 17 52 656173 6 3 1 GalNAc₃-1a A_(d) 147 10 3 17 21 6630866 3 1 GalNAc₃-3a A_(d) 155 10 2 17 9 678347 6 3 1 GalNAc₃-7a A_(d) 15510 1 17 8 678348 6 3 1 GalNAc₃-10a A_(d) 155 10 1 17 6 678349 6 3 1GalNAc₃-13a A_(d) 155 10 1 17 5

Example 85: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising a GalNAc₃ Conjugate

Oligonucleotides listed in Table 76 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

Treatment

Six to eight week old C57BL/6 mice were each injected subcutaneouslyonce per week at a dosage shown below, for a total of three doses, withan oligonucleotide listed in Table 76 or with saline. Each treatmentgroup consisted of 4 animals. The mice were sacrificed 48 hoursfollowing the final administration to determine the SRB-1 mRNA levelsusing real-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. The resultsbelow are presented as the average percent of liver SRB-1 mRNA levelsfor each treatment group, normalized to the saline control.

As illustrated in Tables 80 and 81, treatment with antisenseoligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 80 SRB-1 mRNA in liver ISIS Dosage SRB-1 mRNA (% GalNAc₃ No.(mg/kg) Saline) Cluster CM Saline n/a 100 n/a n/a 655861 0.1 94GalNAc₃-1a A_(d) 0.3 119 1 68 3 32 661161 0.1 120 GalNAc₃-3a A_(d) 0.3107 1 68 3 26 666881 0.1 107 GalNAc₃-10a A_(d) 0.3 107 1 69 3 27 6669810.1 120 GalNAc₃-7a A_(d) 0.3 103 1 54 3 21 670061 0.1 118 GalNAc₃-13aA_(d) 0.3 89 1 52 3 18 677842 0.1 119 GalNAc₃-20a A_(d) 0.3 96 1 65 3 23

TABLE 81 SRB-1 mRNA in liver ISIS Dosage SRB-1 mRNA (% GalNAc₃ No.(mg/kg) Saline) Cluster CM 661161 0.1 107 GalNAc₃-3a A_(d) 0.3 95 1 53 318 677841 0.1 110 GalNAc₃-19a A_(d) 0.3 88 1 52 3 25

Liver transaminase levels, total bilirubin, BUN, and body weights werealso measured using standard protocols. Average values for eachtreatment group are shown in Table 82 below.

TABLE 82 ISIS Dosage ALT AST Bilirubin BUN Body Weight No. (mg/kg) (U/L)(U/L) (mg/dL) (mg/dL) (% baseline) GalNAc₃ Cluster CM Saline n/a 19 390.17 26 118 n/a n/a 655861 0.1 25 47 0.17 27 114 GalNAc₃-1a A_(d) 0.3 2956 0.15 27 118 1 20 32 0.14 24 112 3 27 54 0.14 24 115 661161 0.1 35 830.13 24 113 GalNAc₃-3a A_(d) 0.3 42 61 0.15 23 117 1 34 60 0.18 22 116 329 52 0.13 25 117 666881 0.1 30 51 0.15 23 118 GalNAc₃-10a A_(d) 0.3 4982 0.16 25 119 1 23 45 0.14 24 117 3 20 38 0.15 21 112 666981 0.1 21 410.14 22 113 GalNAc₃-7a A_(d) 0.3 29 49 0.16 24 112 1 19 34 0.15 22 111 377 78 0.18 25 115 670061 0.1 20 63 0.18 24 111 GalNAc₃-13a A_(d) 0.3 2057 0.15 21 115 1 20 35 0.14 20 115 3 27 42 0.12 20 116 677842 0.1 20 380.17 24 114 GalNAc₃-20a A_(d) 0.3 31 46 0.17 21 117 1 22 34 0.15 21 1193 41 57 0.14 23 118

Example 86: Antisense Inhibition In Vivo by Oligonucleotides TargetingTTR Comprising a GalNAc₃ Cluster

Oligonucleotides listed in Table 83 below were tested in adose-dependent study for antisense inhibition of human transthyretin(TTR) in transgenic mice that express the human TTR gene.

Treatment

Eight week old TTR transgenic mice were each injected subcutaneouslyonce per week for three weeks, for a total of three doses, with anoligonucleotide and dosage listed in the tables below or with PBS. Eachtreatment group consisted of 4 animals. The mice were sacrificed 72hours following the final administration. Tail bleeds were performed atvarious time points throughout the experiment, and plasma TTR protein,ALT, and AST levels were measured and reported in Tables 85-87. Afterthe animals were sacrificed, plasma ALT, AST, and human TTR levels weremeasured, as were body weights, organ weights, and liver human TTR mRNAlevels. TTR protein levels were measured using a clinical analyzer(AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREEN® RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.) were usedaccording to standard protocols to determine liver human TTR mRNAlevels. The results presented in Tables 84-87 are the average values foreach treatment group. The mRNA levels are the average values relative tothe average for the PBS group. Plasma protein levels are the averagevalues relative to the average value for the PBS group at baseline. Bodyweights are the average percent weight change from baseline untilsacrifice for each individual treatment group. Organ weights shown arenormalized to the animal's body weight, and the average normalized organweight for each treatment group is then presented relative to theaverage normalized organ weight for the PBS group.

In Tables 84-87, “BL” indicates baseline, measurements that were takenjust prior to the first dose. As illustrated in Tables 84 and 85,treatment with antisense oligonucleotides lowered TTR expression levelsin a dose-dependent manner. The oligonucleotides comprising a GalNAcconjugate were more potent than the parent lacking a GalNAc conjugate(ISIS 420915). Furthermore, the oligonucleotides comprising a GalNAcconjugate and mixed PS/PO internucleoside linkages were even more potentthan the oligonucleotide comprising a GalNAc conjugate and full PSlinkages.

TABLE 83 Oligonucleotides targeting human TTR GalNAc SEQ Isis No.Sequence 5′ to 3′ Linkages cluster CM ID No. 420915 T_(es)^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS n/a n/a 156 A_(es)T_(es)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 660261 T_(es)^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS GalNAc₃-1a A_(d) 157A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′)-GalNAc ₃-1 _(a)682883 GalNAc ₃-3 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)PS/PO GalNAc₃-3a PO 156 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682884 GalNAc ₃-7 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)PS/PO GalNAc₃-7a PO 156 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682885 GalNAc ₃-10 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) PS/POGalNAc₃-10a PO 156 A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682886 GalNAc ₃-13 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) PS/POGalNAc₃-13a PO 156 A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 684057 T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS/PO GalNAc₃-19a A_(d) 157A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′)-GalNAc ₃-19 _(a)The legend for Table 85 can be found in Example 74. The structure ofGalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_(a) wasshown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example48. The structure of GalNAc₃-10_(a) was shown in Example 46. Thestructure of GalNAc₃-13_(a) was shown in Example 62. The structure ofGalNAc₃-19_(a) was shown in Example 70.

TABLE 84 Antisense inhibition of human TTR in vivo Isis Dosage TTR mRNA(% Plasma TTR protein GalNAc SEQ No. (mg/kg) PBS) (% PBS) cluster CM IDNo. PBS n/a 100 100 n/a n/a 420915 6 99 95 n/a n/a 156 20 48 65 60 18 28660261 0.6 113 87 GalNAc₃-1a A_(d) 157 2 40 56 6 20 27 20 9 11

TABLE 85 Antisense inhibition of human TTR in vivo TTR Plasma TTRprotein (% PBS at BL) Dosage mRNA Day 17 GalNAc SEQ ID Isis No. (mg/kg)(% PBS) BL Day 3 Day 10 (After sac) cluster CM No. PBS n/a 100 100 96 90114 n/a n/a 420915 6 74 106 86 76 83 n/a n/a 156 20 43 102 66 61 58 6024 92 43 29 32 682883 0.6 60 88 73 63 68 GalNAc₃- PO 156 2 18 75 38 2323 3a 6 10 80 35 11 9 682884 0.6 56 88 78 63 67 GalNAc₃- PO 156 2 19 7644 25 23 7a 6 15 82 35 21 24 682885 0.6 60 92 77 68 76 GalNAc₃- PO 156 222 93 58 32 32 10a 6 17 85 37 25 20 682886 0.6 57 91 70 64 69 GalNAc₃-PO 156 2 21 89 50 31 30 13a 6 18 102 41 24 27 684057 0.6 53 80 69 56 62GalNAc₃- A_(d) 157 2 21 92 55 34 30 19a 6 11 82 50 18 13

TABLE 86 Transaminase levels, body weight changes, and relative organweights ALT (U/L) AST (U/L) SEQ Dosage Day Day Day Day Day Day BodyLiver Spleen Kidney ID Isis No. (mg/kg) BL 3 10 17 BL 3 10 17 (% BL) (%PBS) (% PBS) (% PBS) No. PBS n/a 33 34 33 24 58 62 67 52 105 100 100 100n/a 420915 6 34 33 27 21 64 59 73 47 115 99 89 91 156 20 34 30 28 19 6454 56 42 111 97 83 89 60 34 35 31 24 61 58 71 58 113 102 98 95 6602610.6 33 38 28 26 70 71 63 59 111 96 99 92 157 2 29 32 31 34 61 60 68 61118 100 92 90 6 29 29 28 34 58 59 70 90 114 99 97 95 20 33 32 28 33 6454 68 95 114 101 106 92

TABLE 87 Transaminase levels, body weight changes, and relative organweights ALT (U/L) AST (U/L) SEQ Dosage Day Day Day Day Day Day BodyLiver Spleen Kidney ID Isis No. (mg/kg) BL 3 10 17 BL 3 10 17 (% BL) (%PBS) (% PBS) (% PBS) No. PBS n/a 32 34 37 41 62 78 76 77 104 100 100 100n/a 420915 6 32 30 34 34 61 71 72 66 102 103 102 105 156 20 41 34 37 3380 76 63 54 106 107 135 101 60 36 30 32 34 58 81 57 60 106 105 104 99682883 0.6 32 35 38 40 53 81 74 76 104 101 112 95 156 2 38 39 42 43 7184 70 77 107 98 116 99 6 35 35 41 38 62 79 103 65 105 103 143 97 6828840.6 33 32 35 34 70 74 75 67 101 100 130 00 156 2 31 32 38 38 63 77 66 55104 103 122 100 6 38 32 36 34 65 85 80 62 99 105 129 95 682885 0.6 39 2637 35 63 63 77 59 100 109 109 112 156 2 30 26 38 40 54 56 71 72 102 98111 102 6 27 27 34 35 46 52 56 64 102 98 113 96 682886 0.6 30 40 34 3658 87 54 61 104 90 120 101 156 2 27 26 34 36 51 55 55 69 103 91 105 92 640 28 34 37 107 54 61 69 109 100 102 99 684057 0.6 35 26 33 39 56 51 5169 104 99 110 102 157 2 33 32 31 40 54 57 56 87 103 100 112 97 6 39 3335 40 67 52 55 92 98 104 121 108

Example 87: Duration of Action In Vivo by Single Doses ofOligonucleotides Targeting TTR Comprising a GalNAc₃ Cluster

ISIS numbers 420915 and 660261 (see Table 83) were tested in a singledose study for duration of action in mice. ISIS numbers 420915, 682883,and 682885 (see Table 83) were also tested in a single dose study forduration of action in mice.

Treatment

Eight week old, male transgenic mice that express human TTR were eachinjected subcutaneously once with 100 mg/kg ISIS No. 420915 or 13.5mg/kg ISIS No. 660261. Each treatment group consisted of 4 animals. Tailbleeds were performed before dosing to determine baseline and at days 3,7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels weremeasured as described in Example 86. The results below are presented asthe average percent of plasma TTR levels for each treatment group,normalized to baseline levels.

TABLE 88 Plasma TTR protein levels Time point SEQ ISIS Dosage (days TTRGalNAc₃ ID No. (mg/kg) post-dose) (% baseline) Cluster CM No. 420915 1003 30 n/a n/a 156 1 23 10 35 17 53 24 75 39 100 660261 13.5 3 27GalNAc₃-1a A_(d) 157 7 21 10 22 17 36 24 48 39 69Treatment

Female transgenic mice that express human TTR were each injectedsubcutaneously once with 100 mg/kg ISIS No. 420915, 10.0 mg/kg ISIS No.682883, or 10.0 mg/kg 682885. Each treatment group consisted of 4animals. Tail bleeds were performed before dosing to determine baselineand at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTRprotein levels were measured as described in Example 86. The resultsbelow are presented as the average percent of plasma TTR levels for eachtreatment group, normalized to baseline levels.

TABLE 89 Plasma TTR protein levels Time point SEQ ISIS Dosage (days TTRGalNAc₃ ID No. (mg/kg) post-dose) (% baseline) Cluster CM No. 420915 1003 48 n/a n/a 156 7 48 10 48 17 66 31 80 682883 10.0 3 45 GalNAc₃-3a  PO156 7 37 10 38 17 42 31 65 682885 10.0 3 40 GalNAc₃-10a PO 156 7 33 1034 17 40 31 64The results in Tables 88 and 89 show that the oligonucleotidescomprising a GalNAc conjugate are more potent with a longer duration ofaction than the parent oligonucleotide lacking a conjugate (ISIS420915).

Example 88: Splicing Modulation In Vivo by Oligonucleotides TargetingSMN Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 90 were tested for splicingmodulation of human survival of motor neuron (SMN) in mice.

TABLE 90 Modified ASOs targeting SMN ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 387954 A_(es)T_(es)T_(es) ^(m)C_(es)A_(es)^(m)C_(es)T_(es)T_(es)T_(es)^(m)C_(es)A_(es)T_(es)A_(es)A_(es)T_(es)G_(es) ^(m)C_(es)T_(es)G_(es)n/a n/a 158 G_(e) 699819 GalNAc ₃-7 _(a)-_(o′)A_(es)T_(es)T_(es)^(m)C_(es)A_(es) ^(m)C_(es)T_(es)T_(es)T_(es)^(m)C_(es)A_(es)T_(es)A_(es)A_(es) GalNAc₃-7a PO 158 T_(es)G_(es)^(m)C_(es)T_(es)G_(es)G_(e) 699821 GalNAc ₃-7_(a)-_(o′)A_(es)T_(eo)T_(eo) ^(m)C_(eo)A_(eo)^(m)C_(eo)T_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(eo)T_(eo)A_(eo) GalNAc₃-7a PO158 A_(eo)T_(eo)G_(eo) ^(m)C_(eo)T_(es)G_(es)G_(e) 700000A_(es)T_(es)T_(es) ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)T_(es)T_(es)^(m)C_(es)A_(es)T_(es)A_(es)A_(es)T_(es)G_(es) ^(m)C_(es)T_(es)G_(es)GalNAc₃-1a A_(d) 157 G_(eo) A _(do′)-GalNAc ₃-1 _(a) 703421X-ATT^(m)CA^(m)CTTT^(m)CATAATG^(m)CTGG n/a n/a 158 703422 GalNAc ₃-7_(b)-X-ATT^(m)CA^(m)CTTT^(m)CATAATG^(m)CTGG GalNAc₃-7b n/a 158The structure of GalNAc₃-7_(a) was shown previously in Example 48. “X”indicates a 5′ primary amine generated by Gene Tools (Philomath, Oreg.),and GalNAc₃-7_(b) indicates the structure of GalNAc₃-7_(a) lacking the—NH—C₆—O portion of the linker as shown below:

ISIS numbers 703421 and 703422 are morphlino oligonucleotides, whereineach nucleotide of the two oligonucleotides is a morpholino nucleotide.Treatment

Six week old transgenic mice that express human SMN were injectedsubcutaneously once with an oligonucleotide listed in Table 91 or withsaline. Each treatment group consisted of 2 males and 2 females. Themice were sacrificed 3 days following the dose to determine the liverhuman SMN mRNA levels both with and without exon 7 using real-time PCRaccording to standard protocols. Total RNA was measured using Ribogreenreagent. The SMN mRNA levels were normalized to total mRNA, and furthernormalized to the averages for the saline treatment group. The resultingaverage ratios of SMN mRNA including exon 7 to SMN mRNA missing exon 7are shown in Table 91. The results show that fully modifiedoligonucleotides that modulate splicing and comprise a GalNAc conjugateare significantly more potent in altering splicing in the liver than theparent oligonucleotides lacking a GlaNAc conjugate. Furthermore, thistrend is maintained for multiple modification chemistries, including2′-MOE and morpholino modified oligonucleotides.

TABLE 91 Effect of oligonucleotides targeting human SMN in vivo ISISDose +Exon 7/ GalNAc₃ SEQ No. (mg/kg) −Exon 7 Cluster CM ID No. Salinen/a 1.00 n/a n/a n/a 387954 32 1.65 n/a n/a 158 387954 288 5.00 n/a n/a158 699819 32 7.84 GalNAc₃-7a PO 158 699821 32 7.22 GalNAc₃-7a PO 158700000 32 6.91 GalNAc₃-1a A_(d) 159 703421 32 1.27 n/a n/a 158 703422 324.12 GalNAc₃-7b n/a 158

Example 89: Antisense Inhibition In Vivo by Oligonucleotides TargetingApolipoprotein A (Apo(a)) Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 92 below were tested in a study fordose-dependent inhibition of Apo(a) in transgenic mice.

TABLE 92 Modified ASOs targeting Apo(a) ISIS GalNAc₃ SEQ ID No.Sequences (5′ to 3′) Cluster CM No. 494372 T_(es)G_(es) ^(m)C_(es)T_(es)^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds) n/a n/a 58 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681257GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 58 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)The structure of GalNAc₃-7_(a) was shown in Example 48.Treatment

Eight week old, female C57BL/6 mice (Jackson Laboratory, Bar Harbor,Me.) were each injected subcutaneously once per week at a dosage shownbelow, for a total of six doses, with an oligonucleotide listed in Table92 or with PBS. Each treatment group consisted of 3-4 animals. Tailbleeds were performed the day before the first dose and weekly followingeach dose to determine plasma Apo(a) protein levels. The mice weresacrificed two days following the final administration. Apo(a) livermRNA levels were determined using real-time PCR and RIBOGREEN® RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.) accordingto standard protocols. Apo(a) plasma protein levels were determinedusing ELISA, and liver transaminase levels were determined. The mRNA andplasma protein results in Table 93 are presented as the treatment groupaverage percent relative to the PBS treated group. Plasma protein levelswere further normalized to the baseline (BL) value for the PBS group.Average absolute transaminase levels and body weights (% relative tobaseline averages) are reported in Table 94.

As illustrated in Table 93, treatment with the oligonucleotides loweredApo(a) liver mRNA and plasma protein levels in a dose-dependent manner.Furthermore, the oligonucleotide comprising the GalNAc conjugate wassignificantly more potent with a longer duration of action than theparent oligonucleotide lacking a GalNAc conjugate. As illustrated inTable 94, transaminase levels and body weights were unaffected by theoligonucleotides, indicating that the oligonucleotides were welltolerated.

TABLE 93 Apo(a) liver mRNA and plasma protein levels Apo(a) Dosage mRNAApo(a) plasma protein (% PBS) ISIS (mg/ (% Week Week Week Week Week WeekNo. kg) PBS) BL 1 2 3 4 5 6 PBS n/a 100 100 120 119 113 88 121 97 4943723 80 84 89 91 98 87 87 79 10 30 87 72 76 71 57 59 46 30 5 92 54 28 10 79 7 681257 0.3 75 79 76 89 98 71 94 78 1 19 79 88 66 60 54 32 24 3 2 8252 17 7 4 6 5 10 2 79 17 6 3 2 4 5

TABLE 94 ISIS Dosage ALT AST Body weight No. (mg/kg) (U/L) (U/L) (%baseline) PBS n/a 37 54 103 494372 3 28 68 106 10 22 55 102 30 19 48 103681257 0.3 30 80 104 1 26 47 105 3 29 62 102 10 21 52 107

Example 90: Antisense Inhibition In Vivo by Oligonucleotides TargetingTTR Comprising a GalNAc₃ Cluster

Oligonucleotides listed in Table 95 below were tested in adose-dependent study for antisense inhibition of human transthyretin(TTR) in transgenic mice that express the human TTR gene.

Treatment

TTR transgenic mice were each injected subcutaneously once per week forthree weeks, for a total of three doses, with an oligonucleotide anddosage listed in Table 96 or with PBS. Each treatment group consisted of4 animals. Prior to the first dose, a tail bleed was performed todetermine plasma TTR protein levels at baseline (BL). The mice weresacrificed 72 hours following the final administration. TTR proteinlevels were measured using a clinical analyzer (AU480, Beckman Coulter,CA). Real-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) were used according to standard protocols todetermine liver human TTR mRNA levels. The results presented in Table 96are the average values for each treatment group. The mRNA levels are theaverage values relative to the average for the PBS group. Plasma proteinlevels are the average values relative to the average value for the PBSgroup at baseline. “BL” indicates baseline, measurements that were takenjust prior to the first dose. As illustrated in Table 96, treatment withantisense oligonucleotides lowered TTR expression levels in adose-dependent manner. The oligonucleotides comprising a GalNAcconjugate were more potent than the parent lacking a GalNAc conjugate(ISIS 420915), and oligonucleotides comprising a phosphodiester ordeoxyadenosine cleavable moiety showed significant improvements inpotency compared to the parent lacking a conjugate (see ISIS numbers682883 and 666943 vs 420915 and see Examples 86 and 87).

TABLE 95 Oligonucleotides targeting human TTR GalNAc SEQ Isis No.Sequence 5′ to 3′ Linkages cluster CM ID No. 420915 T_(es)^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS n/a n/a 156 A_(es)T_(es)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682883 GalNAc ₃ -3 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)PS/PO GalNAc₃-3a PO 156 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 666943 GalNAc ₃ -3 _(a-o′) A _(do)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-3aA_(d) 160 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682887 GalNAc ₃ -7 _(a-o′) A _(do)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-7aA_(d) 160 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682888 GalNAc ₃ -10 _(a-o′) A_(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/POGalNAc₃-10a A_(d) 160^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682889 GalNAc ₃ -13 _(a-o′) A _(do)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-13aA_(d) 160 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo)^(m)C_(es) ^(m)C_(es) ^(m)C_(e)The legend for Table 95 can be found in Example 74. The structure ofGalNAc₃-3_(a) was shown in Example 39. The structure of GalNAc₃-7_(a)was shown in Example 48. The structure of GalNAc₃-10_(a) was shown inExample 46. The structure of GalNAc₃-13_(a) was shown in Example 62.

TABLE 96 Antisense inhibition of human TTR in vivo Dosage TTR mRNA TTRprotein GalNAc Isis No. (mg/kg) (% PBS) (% BL) cluster CM PBS n/a 100124 n/a n/a 420915 6 69 114 n/a n/a 20 71 86 60 21 36 682883 0.6 61 73GalNAc₃-3a PO 2 23 36 6 18 23 666943 0.6 74 93 GalNAc₃-3a A_(d) 2 33 576 17 22 682887 0.6 60 97 GalNAc₃-7a A_(d) 2 36 49 6 12 19 682888 0.6 6592 GalNAc₃-10a A_(d) 2 32 46 6 17 22 682889 0.6 72 74 GalNAc₃-13a A_(d)2 38 45 6 16 18

Example 91: Antisense Inhibition In Vivo by Oligonucleotides TargetingFactor VII Comprising a GalNAc₃ Conjugate in Non-Human Primates

Oligonucleotides listed in Table 97 below were tested in a non-terminal,dose escalation study for antisense inhibition of Factor VII in monkeys.

Treatment

Non-naïve monkeys were each injected subcutaneously on days 0, 15, and29 with escalating doses of an oligonucleotide listed in Table 97 orwith PBS. Each treatment group consisted of 4 males and 1 female. Priorto the first dose and at various time points thereafter, blood drawswere performed to determine plasma Factor VII protein levels. Factor VIIprotein levels were measured by ELISA. The results presented in Table 98are the average values for each treatment group relative to the averagevalue for the PBS group at baseline (BL), the measurements taken justprior to the first dose. As illustrated in Table 98, treatment withantisense oligonucleotides lowered Factor VII expression levels in adose-dependent manner, and the oligonucleotide comprising the GalNAcconjugate was significantly more potent in monkeys compared to theoligonucleotide lacking a GalNAc conjugate.

TABLE 97 Oligonucleotides targeting Factor VII GalNAc SEQ Isis No.Sequence 5′ to 3′ Linkages cluster CM ID No. 407935 A_(es)T_(es)G_(es)^(m)C_(es)A_(es)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds) PS n/a n/a 161 T_(es) ^(m)C_(es)T_(es)G_(es)A_(e)686892 GalNAc ₃ -10 _(a-o′)A_(es)T_(es)G_(es)^(m)C_(es)A_(es)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) PS GalNAc₃-10a PO 161A_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)T_(es)G_(es)A_(e)The legend for Table 97 can be found in Example 74. The structure ofGalNAc₃-10_(a) was shown in Example 46.

TABLE 98 Factor VII plasma protein levels ISIS No. Day Dose (mg/kg)Factor VII (% BL) 407935 0 n/a 100 15 10 87 22 n/a 92 29 30 77 36 n/a 4643 n/a 43 686892 0  3 100 15 10 56 22 n/a 29 29 30 19 36 n/a 15 43 n/a11

Example 92: Antisense Inhibition in Primary Hepatocytes by AntisenseOligonucleotides Targeting ApoCIII Comprising a GalNAc₃ Conjugate

Primary mouse hepatocytes were seeded in 96-well plates at 15,000 cellsper well, and the oligonucleotides listed in Table 99, targeting mouseApoC-III, were added at 0.46, 1.37, 4.12, or 12.35, 37.04, 111.11, or333.33 nM or 1.00 μM. After incubation with the oligonucleotides for 24hours, the cells were lysed and total RNA was purified using RNeasy(Qiagen). ApoC-III mRNA levels were determined using real-time PCR andRIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.) accordingto standard protocols. IC₅₀ values were determined using Prism 4software (GraphPad). The results show that regardless of whether thecleavable moiety was a phosphodiester or a phosphodiester-linkeddeoxyadensoine, the oligonucleotides comprising a GalNAc conjugate weresignificantly more potent than the parent oligonucleotide lacking aconjugate.

TABLE 99 Inhibition of mouse APOC-III expression in mouse primaryhepatocytes ISIS IC₅₀ SEQ No. Sequence (5′ to 3′) CM (nM) ID No. 440670^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) n/a 13.20 162 661180^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) A_(d) 1.40 163 A_(es)G_(es) ^(m)C_(es)A_(eo) A _(do′) -GalNAc₃ -1 _(a) 680771 GalNAc ₃ -3 _(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 0.70 162 A_(es)G_(es) ^(m)C_(es)A_(e) 680772 GalNAc ₃ -7_(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 1.70 162 A_(es)G_(es) ^(m)C_(es)A_(e) 680773 GalNAc ₃ -10_(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 2.00 162 A_(es)G_(es) ^(m)C_(es)A_(e) 680774 GalNAc ₃ -13_(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 1.50 162 A_(es)G_(es) ^(m)C_(es)A_(e) 681272 GalNAc ₃ -3_(a-o′) ^(m)C_(es)A_(eo)G_(eo)^(m)C_(eo)T_(eo)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(eo) PO <0.46 162 A_(eo)G_(es) ^(m)C_(es)A_(e) 681273 GalNAc ₃ -3_(a) - _(o′) A _(do) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)A_(d) 1.10 164 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 683733^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) A_(d) 2.50 163 A_(es)G_(es) ^(m)C_(es)A_(eo) A _(do′) -GalNAc₃ -19 _(a)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, GalNAc₃-13_(a) wasshown in Example 62, and GalNAc₃-19_(a) was shown in Example 70.

Example 93: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising Mixed Wings and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 100 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

TABLE 100 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 449093 T_(ks)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds) A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) n/a n/a 165 699806 GalNAc ₃-3 _(a) - _(o′)T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-3a PO 165 T_(ds)T_(ks)^(m)C_(ks) ^(m)C_(k) 699807 GalNAc ₃ -7 _(a) - _(o′)T_(ks)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds)A_(ds)^(m)C_(ds) GalNAc₃-7a PO 165 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 699809GalNAc ₃ -7 _(a) - _(o′)T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds) A_(ds)T_(ds) G_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 165T_(ds)T_(es) ^(m)C_(es) ^(m)C_(e) 699811 GalNAc ₃ -7 _(a) -_(o′)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 165 T_(ds)T_(ks) ^(m)C_(ks)^(m)C_(k) 699813 GalNAc ₃ -7 _(a) - _(o′)T_(ks)T_(ds)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds)A_(ds)^(m)C_(ds) GalNAc₃-7a PO 165 T_(ds)T_(ks) ^(m)C_(ds) ^(m)C_(k) 699815GalNAc ₃ -7 _(a) - _(o′)T_(es)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds) A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 165T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(e)The structure of GalNAc₃-3_(a) was shown previously in Example 39, andthe structure of GalNAc₃-7a was shown previously in Example 48.Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside(cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o”indicates phosphodiester internucleoside linkages (PO). Superscript “m”indicates 5-methylcytosines.Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once at the dosage shown below with anoligonucleotide listed in Table 100 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration. Liver SRB-1 mRNA levels were measured usingreal-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNAlevels according to standard protocols. The results are presented as theaverage percent of SRB-1 mRNA levels for each treatment group relativeto the saline control group. As illustrated in Table 101, treatment withantisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependentmanner, and the gapmer oligonucleotides comprising a GalNAc conjugateand having wings that were either full cEt or mixed sugar modificationswere significantly more potent than the parent oligonucleotide lacking aconjugate and comprising full cEt modified wings.

Body weights, liver transaminases, total bilirubin, and BUN were alsomeasured, and the average values for each treatment group are shown inTable 101. Body weight is shown as the average percent body weightrelative to the baseline body weight (% BL) measured just prior to theoligonucleotide dose.

TABLE 101 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and bodyweights SRB-1 Body ISIS Dosage mRNA ALT AST weight No. (mg/kg) (% PBS)(U/L) (U/L) Bil BUN (% BL) PBS n/a 100 31 84 0.15 28 102 449093 1 111 1848 0.17 31 104 3 94 20 43 0.15 26 103 10 36 19 50 0.12 29 104 699806 0.1114 23 58 0.13 26 107 0.3 59 21 45 0.12 27 108 1 25 30 61 0.12 30 104699807 0.1 121 19 41 0.14 25 100 0.3 73 23 56 0.13 26 105 1 24 22 690.14 25 102 699809 0.1 125 23 57 0.14 26 104 0.3 70 20 49 0.10 25 105 133 34 62 0.17 25 107 699811 0.1 123 48 77 0.14 24 106 0.3 94 20 45 0.1325 101 1 66 57 104 0.14 24 107 699813 0.1 95 20 58 0.13 28 104 0.3 98 2261 0.17 28 105 1 49 19 47 0.11 27 106 699815 0.1 93 30 79 0.17 25 1050.3 64 30 61 0.12 26 105 1 24 18 41 0.14 25 106

Example 94: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising 2′-Sugar Modifications and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 102 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

TABLE 102 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es) n/a n/a 143 T_(es)T_(e)700989 G_(ms)C_(ms)U_(ms)U_(ms)C_(ms)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)U_(ms)C_(ms)C_(ms)n/a n/a 166 U_(ms)U_(m) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₃-3a PO 143^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 700991 GalNAc ₃-7 _(a) - _(o′)G_(ms)C_(ms)U_(ms)U_(ms)C_(ms)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds) GalNAc₃-7a PO 166 A_(ds)^(m)C_(ds)T_(ds)U_(ms)C_(ms)C_(ms)U_(ms)U_(m)Subscript “m” indicates a 2′-O-methyl modified nucleoside. See Example74 for complete table legend. The structure of GalNAc₃-3_(a) was shownpreviously in Example 39, and the structure of GalNAc₃-7a was shownpreviously in Example 48.Treatment

The study was completed using the protocol described in Example 93.Results are shown in Table 103 below and show that both the 2′-MOE and2′-OMe modified oligonucleotides comprising a GalNAc conjugate weresignificantly more potent than the respective parent oligonucleotideslacking a conjugate. The results of the body weights, livertransaminases, total bilirubin, and BUN measurements indicated that thecompounds were all well tolerated.

TABLE 103 SRB-1 mRNA ISIS No. Dosage (mg/kg) SRB-1 mRNA (% PBS) PBS n/a100 353382 5 116 15 58 45 27 700989 5 120 15 92 45 46 666904 1 98 3 4510 17 700991 1 118 3 63 10 14

Example 95: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising Bicyclic Nucleosides and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 104 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

TABLE 104 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/an/a 137 666905 GalNAc ₃ -3 _(a) - _(o′)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) GalNAc₃-3_(a) PO 137 699782 GalNAc ₃ -7_(a) - _(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)GalNAc₃-7_(a) PO 137 699783 GalNAc ₃ -3 _(a) - _(o′)T_(ls)^(m)C_(ls)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ls) ^(m)C_(l) GalNAc₃-3_(a) PO 137 653621 T_(ls)^(m)C_(ls)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ls) ^(m)C_(lo) A _(do′) -GalNAc ₃ -1 _(a)GalNAc₃-1_(a) A_(d) 138 439879 T_(gs) ^(m)C_(gs)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(gs) ^(m)C_(g) n/an/a 137 699789 GalNAc ₃ -3 _(a) - _(o′)T_(gs)^(m)C_(gs)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(gs) ^(m)C_(g) GalNAc₃-3_(a) PO 137Subscript “g” indicates a fluoro-HNA nucleoside, subscript “1” indicatesa locked nucleoside comprising a 2′-O—CH₂-4′ bridge. See the Example 74table legend for other abbreviations. The structure of GalNAc₃-1 a wasshown previously in Example 9, the structure of GalNAc₃-3_(a) was shownpreviously in Example 39, and the structure of GalNAc₃-7a was shownpreviously in Example 48.Treatment

The study was completed using the protocol described in Example 93.Results are shown in Table 105 below and show that oligonucleotidescomprising a GalNAc conjugate and various bicyclic nucleosidemodifications were significantly more potent than the parentoligonucleotide lacking a conjugate and comprising bicyclic nucleosidemodifications. Furthermore, the oligonucleotide comprising a GalNAcconjugate and fluoro-HNA modifications was significantly more potentthan the parent lacking a conjugate and comprising fluoro-HNAmodifications. The results of the body weights, liver transaminases,total bilirubin, and BUN measurements indicated that the compounds wereall well tolerated.

TABLE 105 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and bodyweights ISIS No. Dosage (mg/kg) SRB-1 mRNA (% PBS) PBS n/a 100 440762 1104 3 65 10 35 666905 0.1 105 0.3 56 1 18 699782 0.1 93 0.3 63 1 15699783 0.1 105 0.3 53 1 12 653621 0.1 109 0.3 82 1 27 439879 1 96 3 7710 37 699789 0.1 82 0.3 69 1 26

Example 96: Plasma Protein Binding of Antisense OligonucleotidesComprising a GalNAc₃ Conjugate Group

Oligonucleotides listed in Table 70 targeting ApoC-III andoligonucleotides in Table 106 targeting Apo(a) were tested in anultra-filtration assay in order to assess plasma protein binding.

TABLE 106 Modified oligonucleotides targeting Apo(a) ISIS GalNAc₃ SEQNo. Sequences (5′ to 3′) Cluster CM ID No 494372 T_(es)G_(es)^(m)C_(es)T_(es) ^(m)C_(es)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es) n/a n/a 58 T_(es) ^(m)C_(e) 693401T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es) n/a n/a 58 T_(es) ^(m)C_(e) 681251GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)GalNAc₃-7_(a) PO 58 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681257GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)GalNAc₃-7_(a) PO 58 T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)See the Example 74 for table legend. The structure of GalNAc₃-7a wasshown previously in Example 48.

Ultrafree-MC ultrafiltration units (30,000 NMWL, low-binding regeneratedcellulose membrane, Millipore, Bedford, Mass.) were pre-conditioned with300 μL of 0.5% Tween 80 and centrifuged at 2000 g for 10 minutes, thenwith 3004 of a 300 μg/mL solution of a control oligonucleotide in H₂Oand centrifuged at 2000 g for 16 minutes. In order to assessnon-specific binding to the filters of each test oligonucleotide fromTables 70 and 106 to be used in the studies, 300 μL of a 250 ng/mLsolution of oligonucleotide in H₂O at pH 7.4 was placed in thepre-conditioned filters and centrifuged at 2000 g for 16 minutes. Theunfiltered and filtered samples were analyzed by an ELISA assay todetermine the oligonucleotide concentrations. Three replicates were usedto obtain an average concentration for each sample. The averageconcentration of the filtered sample relative to the unfiltered sampleis used to determine the percent of oligonucleotide that is recoveredthrough the filter in the absence of plasma (% recovery).

Frozen whole plasma samples collected in K3-EDTA from normal, drug-freehuman volunteers, cynomolgus monkeys, and CD-1 mice, were purchased fromBioreclamation LLC (Westbury, N.Y.). The test oligonucleotides wereadded to 1.2 mL aliquots of plasma at two concentrations (5 and 150μg/mL). An aliquot (300 μL) of each spiked plasma sample was placed in apre-conditioned filter unit and incubated at 37° C. for 30 minutes,immediately followed by centrifugation at 2000 g for 16 minutes.Aliquots of filtered and unfiltered spiked plasma samples were analyzedby an ELISA to determine the oligonucleotide concentration in eachsample. Three replicates per concentration were used to determine theaverage percentage of bound and unbound oligonucleotide in each sample.The average concentration of the filtered sample relative to theconcentration of the unfiltered sample is used to determine the percentof oligonucleotide in the plasma that is not bound to plasma proteins (%unbound). The final unbound oligonucleotide values are corrected fornon-specific binding by dividing the % unbound by the % recovery foreach oligonucleotide. The final % bound oligonucleotide values aredetermined by subtracting the final % unbound values from 100. Theresults are shown in Table 107 for the two concentrations ofoligonucleotide tested (5 and 150 μg/mL) in each species of plasma. Theresults show that GalNAc conjugate groups do not have a significantimpact on plasma protein binding. Furthermore, oligonucleotides withfull PS internucleoside linkages and mixed PO/PS linkages both bindplasma proteins, and those with full PS linkages bind plasma proteins toa somewhat greater extent than those with mixed PO/PS linkages.

TABLE 107 Percent of modified oligonucleotide bound to plasma proteinsISIS Human plasma Monkey plasma Mouse plasma No. 5 μg/mL 150 μg/mL 5μg/mL 150 μg/mL 5 μg/mL 150 μg/mL 304801 99.2 98.0 99.8 99.5 98.1 97.2663083 97.8 90.9 99.3 99.3 96.5 93.0 674450 96.2 97.0 98.6 94.4 94.689.3 494372 94.1 89.3 98.9 97.5 97.2 93.6 693401 93.6 89.9 96.7 92.094.6 90.2 681251 95.4 93.9 99.1 98.2 97.8 96.1 681257 93.4 90.5 97.693.7 95.6 92.7

Example 97: Modified Oligonucleotides Targeting TTR Comprising a GalNAc₃Conjugate Group

The oligonucleotides shown in Table 108 comprising a GalNAc conjugatewere designed to target TTR.

TABLE 108 Modified oligonucleotides targeting TTR GalNAc₃ SEQ ID ISISNo. Sequences (5′ to 3′) Cluster CM No 666941 GalNAc ₃ -3 _(a-o′)A_(do)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds)^(m)C_(ds) GalNAc₃-3 A_(d) 160 A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 666942 T_(es) ^(m)C_(eo) T_(eo)T_(eo) G_(eo) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) T_(ds)G_(ds) A_(ds) A_(ds) GalNAc₃-1 A_(d) 157 A_(eo) T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -3 _(a) 682876 GalNAc ₃ -3_(a-o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds)A_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₃-3 PO 156 G_(ds) A_(ds) A_(ds)A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682877 GalNAc ₃ -7_(a-o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds)A_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₃-7 PO 156 G_(ds) A_(ds) A_(ds)A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682878 GalNAc ₃ -10_(a-o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds)A_(ds) ^(m)C_(ds) A_(ds) GalNAc₃-10 PO 156 T_(ds) G_(ds) A_(ds) A_(ds)A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682879 GalNAc ₃ -13_(a-o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds)A_(ds) ^(m)C_(ds) A_(ds) GalNAc₃-13 PO 156 T_(ds) G_(ds) A_(ds) A_(ds)A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682880 GalNAc ₃ -7 _(a-o′)A _(do) T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7 A_(d) 160 A_(ds) T_(ds) G_(ds) A_(ds) A_(ds)A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682881 GalNAc ₃ -10_(a-o′) A _(do) T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds)T_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-10 A_(d) 160 A_(ds) T_(ds) G_(ds)A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682882GalNAc ₃ -13 _(a-o′) A _(do) T_(es) ^(m)C_(es) T_(es) T_(es) G_(es)G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-13 A_(d) 160 A_(ds)T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(e) 684056 T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds)T_(ds) A_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) GalNAc₃-19A_(d) 157 A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′)-GalNAc ₃ -19 _(a)The legend for Table 108 can be found in Example 74. The structure ofGalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_(a) wasshown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example48. The structure of GalNAc₃-10_(a) was shown in Example 46. Thestructure of GalNAc₃-13_(a) was shown in Example 62. The structure ofGalNAc₃-19_(a) was shown in Example 70.

Example 98: Evaluation of Pro-Inflammatory Effects of OligonucleotidesComprising a GalNAc Conjugate in hPMBC Assay

The oligonucleotides listed in Table 109 and were tested forpro-inflammatory effects in an hPMBC assay as described in Examples 23and 24. (See Tables 30, 83, 95, and 108 for descriptions of theoligonucleotides.) ISIS 353512 is a high responder used as a positivecontrol, and the other oligonucleotides are described in Tables 83, 95,and 108. The results shown in Table 109 were obtained using blood fromone volunteer donor. The results show that the oligonucleotidescomprising mixed PO/PS internucleoside linkages produced significantlylower pro-inflammatory responses compared to the same oligonucleotideshaving full PS linkages. Furthermore, the GalNAc conjugate group did nothave a significant effect in this assay.

TABLE 109 ISIS No. E_(max)/EC₅₀ GalNAc₃ cluster Linkages CM 353512 3630n/a PS n/a 420915 802 n/a PS n/a 682881 1311 GalNAc₃-10 PS A_(d) 6828880.26 GalNAc₃-10 PO/PS A_(d) 684057 1.03 GalNAc₃-19 PO/PS A_(d)

Example 99: Binding Affinities of Oligonucleotides Comprising a GalNAcConjugate for the Asialoglycoprotein Receptor

The binding affinities of the oligonucleotides listed in Table 110 (seeTable 76 for descriptions of the oligonucleotides) for theasialoglycoprotein receptor were tested in a competitive receptorbinding assay. The competitor ligand, α1-acid glycoprotein (AGP), wasincubated in 50 mM sodium acetate buffer (pH 5) with 1 Uneuraminidase-agarose for 16 hours at 37° C., and >90% desialylation wasconfirmed by either sialic acid assay or size exclusion chromatography(SEC). Iodine monochloride was used to iodinate the AGP according to theprocedure by Atsma et al. (see J Lipid Res. 1991 January; 32(1):173-81.)In this method, desialylated α1-acid glycoprotein (de-AGP) was added to10 mM iodine chloride, Na¹²⁵I, and 1 M glycine in 0.25 M Na₀H. Afterincubation for 10 minutes at room temperature, ¹²⁵I-labeled de-AGP wasseparated from free ¹²⁵I by concentrating the mixture twice utilizing a3 KDMWCO spin column. The protein was tested for labeling efficiency andpurity on a HPLC system equipped with an Agilent SEC-3 column (7.8×300mm) and a β-RAM counter. Competition experiments utilizing ¹²⁵I-labeledde-AGP and various GalNAc-cluster containing ASOs were performed asfollows. Human HepG2 cells (10⁶ cells/ml) were plated on 6-well platesin 2 ml of appropriate growth media. MEM media supplemented with 10%fetal bovine serum (FBS), 2 mM L-Glutamine and 10 mM HEPES was used.Cells were incubated 16-20 hours @ 37° C. with 5% and 10% CO₂respectively. Cells were washed with media without FBS prior to theexperiment. Cells were incubated for 30 min @37° C. with 1 mlcompetition mix containing appropriate growth media with 2% FBS, 10⁻⁸ M¹²⁵I-labeled de-AGP and GalNAc-cluster containing ASOs at concentrationsranging from 10⁻¹¹ to 10⁻⁵ M. Non-specific binding was determined in thepresence of 10⁻² M GalNAc sugar. Cells were washed twice with mediawithout FBS to remove unbound ¹²⁵I-labeled de-AGP and competitor GalNAcASO. Cells were lysed using Qiagen's RLT buffer containing 1%β-mercaptoethanol. Lysates were transferred to round bottom assay tubesafter a brief 10 min freeze/thaw cycle and assayed on a γ-counter.Non-specific binding was subtracted before dividing ¹²⁵I protein countsby the value of the lowest GalNAc-ASO concentration counts. Theinhibition curves were fitted according to a single site competitionbinding equation using a nonlinear regression algorithm to calculate thebinding affinities (K_(D)'s).

The results in Table 110 were obtained from experiments performed onfive different days. Results for oligonucleotides marked withsuperscript “a” are the average of experiments run on two differentdays. The results show that the oligonucleotides comprising a GalNAcconjugate group on the 5′-end bound the asialoglycoprotein receptor onhuman HepG2 cells with 1.5 to 16-fold greater affinity than theoligonucleotides comprising a GalNAc conjugate group on the 3′-end.

TABLE 110 Asialoglycoprotein receptor binding assay resultsOligonucleotide end to ISIS GalNAc which GalNAc K_(D) No. conjugateconjugate is attached (nM) 661161a GalNAc₃-3 5′ 3.7 666881a GalNAc₃-105′ 7.6 666981 GalNAc₃-7 5′ 6.0 670061 GalNAc₃-13 5′ 7.4 655861aGalNAc₃-1 3′ 11.6 677841a GalNAc₃-19 3′ 60.8

Example 100: Antisense Inhibition In Vivo by Oligonucleotides Comprisinga GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 111a below were tested in a singledose study for duration of action in mice.

TABLE 111a Modified ASOs targeting APO(a) ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 681251 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es)^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)GalNAc₃-7a PO 58 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es)T_(es)^(m)C_(e) 681257 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo)^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 58T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)The structure of GalNAc₃-7_(a) was shown in Example 48.Treatment

Female transgenic mice that express human Apo(a) were each injectedsubcutaneously once per week, for a total of 6 doses, with anoligonucleotide and dosage listed in Table 111b or with PBS. Eachtreatment group consisted of 3 animals. Blood was drawn the day beforedosing to determine baseline levels of Apo(a) protein in plasma and at72 hours, 1 week, and 2 weeks following the first dose. Additional blooddraws will occur at 3 weeks, 4 weeks, 5 weeks, and 6 weeks following thefirst dose. Plasma Apo(a) protein levels were measured using an ELISA.The results in Table 111b are presented as the average percent of plasmaApo(a) protein levels for each treatment group, normalized to baselinelevels (% BL), The results show that the oligonucleotides comprising aGalNAc conjugate group exhibited potent reduction in Apo(a) expression.This potent effect was observed for the oligonucleotide that comprisesfull PS internucleoside linkages and the oligonucleotide that comprisesmixed PO and PS linkages.

TABLE 111b Apo(a) plasma protein levels ISIS Dosage Apo(a) at 72 Apo(a)at 1 Apo(a) at 3 No. (mg/kg) hours (% BL) week (% BL) weeks (% BL) PBSn/a 116 104 107 681251 0.3 97 108 93 1.0 85 77 57 3.0 54 49 11 10.0 2315 4 681257 0.3 114 138 104 1.0 91 98 54 3.0 69 40 6 10.0 30 21 4

Example 101: Antisense Inhibition by Oligonucleotides Comprising aGalNAc Cluster Linked Via a Stable Moiety

The oligonucleotides listed in Table 112 were tested for inhibition ofmouse APOC-III expression in vivo. C57Bl/6 mice were each injectedsubcutaneously once with an oligonucleotide listed in Table 112 or withPBS. Each treatment group consisted of 4 animals. Each mouse treatedwith ISIS 440670 received a dose of 2, 6, 20, or 60 mg/kg. Each mousetreated with ISIS 680772 or 696847 received 0.6, 2, 6, or 20 mg/kg. TheGalNAc conjugate group of ISIS 696847 is linked via a stable moiety, aphosphorothioate linkage instead of a readily cleavable phosphodiestercontaining linkage. The animals were sacrificed 72 hours after the dose.Liver APOC-III mRNA levels were measured using real-time PCR. APOC-IIImRNA levels were normalized to cyclophilin mRNA levels according tostandard protocols. The results are presented in Table 112 as theaverage percent of APOC-III mRNA levels for each treatment grouprelative to the saline control group. The results show that theoligonucleotides comprising a GalNAc conjugate group were significantlymore potent than the oligonucleotide lacking a conjugate group.Furthermore, the oligonucleotide comprising a GalNAc conjugate grouplinked to the oligonucleotide via a cleavable moiety (ISIS 680772) waseven more potent than the oligonucleotide comprising a GalNAc conjugategroup linked to the oligonucleotide via a stable moiety (ISIS 696847).

TABLE 112 Modified oligonucleotides targeting mouse APOC-III APOC-IIIISIS Dosage mRNA (% SEQ No. Sequences (5′ to 3′) CM (mg/kg) PBS) ID No.440670 ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds) n/a 2 92 162G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(es)G_(es) ^(m)C_(es)A_(e) 6 86 2059 60 37 680772 GalNAc ₃ -7 _(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds) PO 0.6 79 162T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(es)G_(es)^(m)C_(es)A_(e) 2 58 6 31 20 13 696847 GalNAc ₃ -7 _(a-s′)^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds) n/a (PS)0.6 83 162 T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(es)G_(es)^(m)C_(es)A_(e) 2 73 6 40 20 28The structure of GalNAc₃-7_(a) was shown in Example 48.

Example 102: Distribution in Liver of Antisense OligonucleotidesComprising a GalNAc Conjugate

The liver distribution of ISIS 353382 (see Table 36) that does notcomprise a GalNAc conjugate and ISIS 655861 (see Table 36) that doescomprise a GalNAc conjugate was evaluated. Male balb/c mice weresubcutaneously injected once with ISIS 353382 or 655861 at a dosagelisted in Table 113. Each treatment group consisted of 3 animals exceptfor the 18 mg/kg group for ISIS 655861, which consisted of 2 animals.The animals were sacrificed 48 hours following the dose to determine theliver distribution of the oligonucleotides. In order to measure thenumber of antisense oligonucleotide molecules per cell, a Ruthenium (II)tris-bipyridine tag (MSD TAG, Meso Scale Discovery) was conjugated to anoligonucleotide probe used to detect the antisense oligonucleotides. Theresults presented in Table 113 are the average concentrations ofoligonucleotide for each treatment group in units of millions ofoligonucleotide molecules per cell. The results show that at equivalentdoses, the oligonucleotide comprising a GalNAc conjugate was present athigher concentrations in the total liver and in hepatocytes than theoligonucleotide that does not comprise a GalNAc conjugate. Furthermore,the oligonucleotide comprising a GalNAc conjugate was present at lowerconcentrations in non-parenchymal liver cells than the oligonucleotidethat does not comprise a GalNAc conjugate. And while the concentrationsof ISIS 655861 in hepatocytes and non-parenchymal liver cells weresimilar per cell, the liver is approximately 80% hepatocytes by volume.Thus, the majority of the ISIS 655861 oligonucleotide that was presentin the liver was found in hepatocytes, whereas the majority of the ISIS353382 oligonucleotide that was present in the liver was found innon-parenchymal liver cells.

TABLE 113 Concentration in whole Concentration in Concentration in non-ISIS Dosage liver (molecules*10{circumflex over ( )}6 hepatocytesparenchymal liver cells No. (mg/kg) per cell) (molecules*10{circumflexover ( )}6 per cell) (molecules*10{circumflex over ( )}6 per cell)353382 3 9.7 1.2 37.2 10 17.3 4.5 34.0 20 23.6 6.6 65.6 30 29.1 11.780.0 60 73.4 14.8 98.0 90 89.6 18.5 119.9 655861 0.5 2.6 2.9 3.2 1 6.27.0 8.8 3 19.1 25.1 28.5 6 44.1 48.7 55.0 18 76.6 82.3 77.1

Example 103: Duration of Action In Vivo of Oligonucleotides TargetingAPOC-III Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 114 below were tested in a singledose study for duration of action in mice.

TABLE 114 Modified ASOs targeting APOC-III ISIS GalNAc₃ SEQ No.Sequences (5′ to 3′) Cluster CM ID No. 304801 A_(es)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) n/a n/a 135T_(es)A_(es)T_(e) 663084 GalNAc ₃ -3 _(a) - _(o′)A_(do)A_(es)G_(eo)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)GalNAc₃-3a A_(d) 151 ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(e) 679241 A_(es)G_(eo)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo) GalNAc₃-19a A_(d) 136T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -19 _(a)

The structure of GalNAc₃-3_(a) was shown in Example 39, andGalNAc₃-19_(a) was shown in Example 70.

Treatment

Female transgenic mice that express human APOC-III were each injectedsubcutaneously once with an oligonucleotide listed in Table 114 or withPBS. Each treatment group consisted of 3 animals. Blood was drawn beforedosing to determine baseline and at 3, 7, 14, 21, 28, 35, and 42 daysfollowing the dose. Plasma triglyceride and APOC-III protein levels weremeasured as described in Example 20. The results in Table 115 arepresented as the average percent of plasma triglyceride and APOC-IIIlevels for each treatment group, normalized to baseline levels. Acomparison of the results in Table 71 of example 79 with the results inTable 115 below show that oligonucleotides comprising a mixture ofphosphodiester and phosphorothioate internucleoside linkages exhibitedincreased duration of action than equivalent oligonucleotides comprisingonly phosphorothioate internucleoside linkages.

TABLE 115 Plasma triglyceride and APOC-III protein levels in transgenicmice Time point (days Triglycerides APOC-III ISIS Dosage post- (%protein GalNAc₃ No. (mg/kg) dose) baseline) (% baseline) Cluster CM PBSn/a 3 96 101 n/a n/a 7 88 98 14 91 103 21 69 92 28 83 81 35 65 86 42 7288 304801 30 3 42 46 n/a n/a 7 42 51 14 59 69 21 67 81 28 79 76 35 72 9542 82 92 663084 10 3 35 28 GalNAc₃-3a  A_(d) 7 23 24 14 23 26 21 23 2928 30 22 35 32 36 42 37 47 679241 10 3 38 30 GalNAc₃-19a A_(d) 7 31 2814 30 22 21 36 34 28 48 34 35 50 45 42 72 64

Example 104: Synthesis of Oligonucleotides Comprising a 5′-GalNAc₂Conjugate

Compound 120 is commercially available, and the synthesis of compound126 is described in Example 49. Compound 120 (1 g, 2.89 mmol), HBTU(0.39 g, 2.89 mmol), and HOBt (1.64 g, 4.33 mmol) were dissolved in DMF(10 mL. and N,N-diisopropylethylamine (1.75 mL, 10.1 mmol) were added.After about 5 min, aminohexanoic acid benzyl ester (1.36 g, 3.46 mmol)was added to the reaction. After 3 h, the reaction mixture was pouredinto 100 mL of 1 M NaHSO₄ and extracted with 2×50 mL ethyl acetate.Organic layers were combined and washed with 3×40 mL sat NaHCO₃ and 2×brine, dried with Na₂SO₄, filtered and concentrated. The product waspurified by silica gel column chromatography (DCM:EA:Hex, 1:1:1) toyield compound 231. LCMS and NMR were consistent with the structure.Compounds 231 (1.34 g, 2.438 mmol) was dissolved in dichloromethane (10mL) and trifluoracetic acid (10 mL) was added. After stirring at roomtemperature for 2 h, the reaction mixture was concentrated under reducedpressure and co-evaporated with toluene (3×10 mL). The residue was driedunder reduced pressure to yield compound 232 as the trifluoracetatesalt. The synthesis of compound 166 is described in Example 54. Compound166 (3.39 g, 5.40 mmol) was dissolved in DMF (3 mL). A solution ofcompound 232 (1.3 g, 2.25 mmol) was dissolved in DMF (3 mL) andN,N-diisopropylethylamine (1.55 mL) was added. The reaction was stirredat room temperature for 30 minutes, then poured into water (80 mL) andthe aqueous layer was extracted with EtOAc (2×100 mL). The organic phasewas separated and washed with sat. aqueous NaHCO₃ (3×80 mL), 1 M NaHSO₄(3×80 mL) and brine (2×80 mL), then dried (Na₂SO₄), filtered, andconcentrated. The residue was purified by silica gel columnchromatography to yield compound 233. LCMS and NMR were consistent withthe structure. Compound 233 (0.59 g, 0.48 mmol) was dissolved inmethanol (2.2 mL) and ethyl acetate (2.2 mL). Palladium on carbon (10 wt% Pd/C, wet, 0.07 g) was added, and the reaction mixture was stirredunder hydrogen atmosphere for 3 h. The reaction mixture was filteredthrough a pad of Celite and concentrated to yield the carboxylic acid.The carboxylic acid (1.32 g, 1.15 mmol, cluster free acid) was dissolvedin DMF (3.2 mL). To this N,N-diisopropylehtylamine (0.3 mL, 1.73 mmol)and PFPTFA (0.30 mL, 1.73 mmol) were added. After 30 min stirring atroom temperature the reaction mixture was poured into water (40 mL) andextracted with EtOAc (2×50 mL). A standard work-up was completed asdescribed above to yield compound 234. LCMS and NMR were consistent withthe structure. Oligonucleotide 235 was prepared using the generalprocedure described in Example 46. The GalNAc₂ cluster portion(GalNAc₂-24_(a)) of the conjugate group GalNAc₂-24 can be combined withany cleavable moiety present on the oligonucleotide to provide a varietyof conjugate groups. The structure of GalNAc₂-24 (GalNAc₂-24_(a)-CM) isshown below:

Example 105: Synthesis of Oligonucleotides Comprising a GalNAc₁-25Conjugate

The synthesis of compound 166 is described in Example 54.Oligonucleotide 236 was prepared using the general procedure describedin Example 46.

Alternatively, oligonucleotide 236 was synthesized using the schemeshown below, and compound 238 was used to form the oligonucleotide 236using procedures described in Example 10.

The GalNAc₁ cluster portion (GalNAc₁-25_(a)) of the conjugate groupGalNAc₁-25 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc₁-25 (GalNAc₁-25_(a)-CM) is shown below:

Example 106: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising a 5′-GalNAc₂ or a 5′-GalNAc₃ Conjugate

Oligonucleotides listed in Tables 116 and 117 were tested indose-dependent studies for antisense inhibition of SRB-1 in mice.

Treatment

Six to week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once with 2, 7, or 20 mg/kg of ISIS No.440762; or with 0.2, 0.6, 2, 6, or 20 mg/kg of ISIS No. 686221, 686222,or 708561; or with saline. Each treatment group consisted of 4 animals.The mice were sacrificed 72 hours following the final administration.Liver SRB-1 mRNA levels were measured using real-time PCR. SRB-1 mRNAlevels were normalized to cyclophilin mRNA levels according to standardprotocols. The antisense oligonucleotides lowered SRB-1 mRNA levels in adose-dependent manner, and the ED₅₀ results are presented in Tables 116and 117. Although previous studies showed that trivalentGalNAc-conjugated oligonucleotides were significantly more potent thandivalent GalNAc-conjugated oligonucleotides, which were in turnsignificantly more potent than monovalent GalNAc conjugatedoligonucleotides (see, e.g., Khorev et al., Bioorg. & Med. Chem., Vol.16, 5216-5231 (2008)), treatment with antisense oligonucleotidescomprising monovalent, divalent, and trivalent GalNAc clusters loweredSRB-1 mRNA levels with similar potencies as shown in Tables 116 and 117.

TABLE 116 Modified oligonucleotides targeting SRB-1 ISIS ED₅₀ SEQ No.Sequences (5′ to 3′) GalNAc Cluster (mg/kg) ID No 440762 T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a 4.7 137 686221 GalNAc ₂ -24 _(a) -_(o′) A _(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₂-24_(a) 0.39 141^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) 686222 GalNAc ₃ -13 _(a) - _(o′) A_(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₃-13_(a) 0.41 141^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)See Example 93 for table legend. The structure of GalNAc₃-13a was shownin Example 62, and the structure of GalNAc₂-24a was shown in Example104.

TABLE 117 Modified oligonucleotides targeting SRB-1 ISIS ED₅₀ SEQ No.Sequences (5′ to 3′) GalNAc Cluster (mg/kg) ID No 440762 T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a 5 137 708561 GalNAc ₁ -25 _(a) -_(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₁-25_(a) 0.4 137^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)See Example 93 for table legend. The structure of GalNAc₁-25a was shownin Example 105.

The concentrations of the oligonucleotides in Tables 116 and 117 inliver were also assessed, using procedures described in Example 75. Theresults shown in Tables 117a and 117b below are the average totalantisense oligonucleotide tissues levels for each treatment group, asmeasured by UV in units of μg oligonucleotide per gram of liver tissue.The results show that the oligonucleotides comprising a GalNAc conjugategroup accumulated in the liver at significantly higher levels than thesame dose of the oligonucleotide lacking a GalNAc conjugate group.Furthermore, the antisense oligonucleotides comprising one, two, orthree GalNAc ligands in their respective conjugate groups allaccumulated in the liver at similar levels. This result is surprising inview of the Khorev et al. literature reference cited above and isconsistent with the activity data shown in Tables 116 and 117 above.

TABLE 117a Liver concentrations of oligonucleotides comprising a GalNAc₂or GalNAc₃ conjugate group Dosage [Antisense GalNAc ISIS No. (mg/kg)oligonucleotide] (μg) cluster CM 440762 2 2.1 n/a n/a 7 13.1 20 31.1686221 0.2 0.9 GalNAc₂-24_(a) A_(d) 0.6 2.7 2 12.0 6 26.5 686222 0.2 0.5GalNAc₃-13_(a) A_(d) 0.6 1.6 2 11.6 6 19.8

TABLE 117b Liver concentrations of oligonucleotides comprising a GalNAc₁conjugate group Dosage [Antisense GalNAc ISIS No. (mg/kg)oligonucleotide] (μg) cluster CM 440762 2 2.3 n/a n/a 7 8.9 20 23.7708561 0.2 0.4 GalNAc₁-25_(a) PO 0.6 1.1 2 5.9 6 23.7 20 53.9

Example 107: Synthesis of Oligonucleotides Comprising a GalNAc₁-26 orGalNAc₁-27 Conjugate

Oligonucleotide 239 is synthesized via coupling of compound 47 (seeExample 15) to acid 64 (see Example 32) using HBTU and DIEA in DMF. Theresulting amide containing compound is phosphitylated, then added to the5′-end of an oligonucleotide using procedures described in Example 10.The GalNAc₁ cluster portion (GalNAc₁-26_(a)) of the conjugate groupGalNAc₁-26 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc₁-26 (GalNAc₁-26_(a)-CM) is shown below:

In order to add the GalNAc₁ conjugate group to the 3′-end of anoligonucleotide, the amide formed from the reaction of compounds 47 and64 is added to a solid support using procedures described in Example 7.The oligonucleotide synthesis is then completed using proceduresdescribed in Example 9 in order to form oligonucleotide 240.

The GalNAc₁ cluster portion (GalNAc₁-27_(a)) of the conjugate groupGalNAc₁-27 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc₁-27 (GalNAc₁-27_(a)-CM) is shown below:

Example 108: Antisense Inhibition In Vivo by Oligonucleotides Comprisinga GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 118 below were tested in a singledose study in mice.

TABLE 118 Modified ASOs targeting APO(a) ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 494372 T_(es)G_(es) ^(m)C_(es)T_(es)^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds) n/a n/a 58 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681251GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 58 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(es)G_(es) T_(es)T_(es) ^(m)C_(e) 681255 GalNAc ₃ -3_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-3a PO 58 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo) T_(es)T_(es) ^(m)C_(e) 681256 GalNAc ₃ -10_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-10a PO 58 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo) T_(es)T_(es) ^(m)C_(e) 681257 GalNAc ₃ -7_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 58 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo) T_(es)T_(es) ^(m)C_(e) 681258 GalNAc ₃ -13_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-13a PO 58 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo) T_(es)T_(es) ^(m)C_(e) 681260 T_(es)G_(eo)^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo) GalNAc₃-19a A_(d) 167T_(es)T_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -19

The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Male transgenic mice that express human Apo(a) were each injectedsubcutaneously once with an oligonucleotide and dosage listed in Table119 or with PBS. Each treatment group consisted of 4 animals. Blood wasdrawn the day before dosing to determine baseline levels of Apo(a)protein in plasma and at 1 week following the first dose. Additionalblood draws will occur weekly for approximately 8 weeks. Plasma Apo(a)protein levels were measured using an ELISA. The results in Table 119are presented as the average percent of plasma Apo(a) protein levels foreach treatment group, normalized to baseline levels (% BL), The resultsshow that the antisense oligonucleotides reduced Apo(a) proteinexpression. Furthermore, the oligonucleotides comprising a GalNAcconjugate group exhibited even more potent reduction in Apo(a)expression than the oligonucleotide that does not comprise a conjugategroup.

TABLE 119 Apo(a) plasma protein levels ISIS Dosage Apo(a) at 1 week No.(mg/kg) (% BL) PBS n/a 143 494372 50 58 681251 10 15 681255 10 14 68125610 17 681257 10 24 681258 10 22 681260 10 26

Example 109: Synthesis of Oligonucleotides Comprising a GalNAc₁-28 orGalNAc₁-29 Conjugate

Oligonucleotide 241 is synthesized using procedures similar to thosedescribed in Example 71 to form the phosphoramidite intermediate,followed by procedures described in Example 10 to synthesize theoligonucleotide. The GalNAc₁ cluster portion (GalNAc₁-28_(a)) of theconjugate group GalNAc₁-28 can be combined with any cleavable moietypresent on the oligonucleotide to provide a variety of conjugate groups.The structure of GalNAc₁-28 (GalNAc₁-28_(a)-CM) is shown below:

In order to add the GalNAc₁ conjugate group to the 3′-end of anoligonucleotide, procedures similar to those described in Example 71 areused to form the hydroxyl intermediate, which is then added to the solidsupport using procedures described in Example 7. The oligonucleotidesynthesis is then completed using procedures described in Example 9 inorder to form oligonucleotide 242.

The GalNAc₁ cluster portion (GalNAc₁-29_(a)) of the conjugate groupGalNAc₁-29 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc₁-29 (GalNAc₁-29_(a)-CM) is shown below:

Example 110: Synthesis of Oligonucleotides Comprising a GalNAc₁-30Conjugate

Oligonucleotide 246 comprising a GalNAc₁-30 conjugate group, wherein Yis selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl,amino, substituted amino, azido, alkenyl or alkynyl, is synthesized asshown above. The GalNAc₁ cluster portion (GalNAc₁-30_(a)) of theconjugate group GalNAc₁-30 can be combined with any cleavable moiety toprovide a variety of conjugate groups. In certain embodiments, Y is partof the cleavable moiety. In certain embodiments, Y is part of a stablemoiety, and the cleavable moiety is present on the oligonucleotide. Thestructure of GalNAc₁-30_(a) is shown below:

Example 111: Synthesis of Oligonucleotides Comprising a GalNAc₂-31 orGalNAc₂-32 Conjugate

Oligonucleotide 250 comprising a GalNAc₂-31 conjugate group, wherein Yis selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl,amino, substituted amino, azido, alkenyl or alkynyl, is synthesized asshown above. The GalNAc₂ cluster portion (GalNAc₂-31_(a)) of theconjugate group GalNAc₂-31 can be combined with any cleavable moiety toprovide a variety of conjugate groups. In certain embodiments, theY-containing group directly adjacent to the 5′-end of theoligonucleotide is part of the cleavable moiety. In certain embodiments,the Y-containing group directly adjacent to the 5′-end of theoligonucleotide is part of a stable moiety, and the cleavable moiety ispresent on the oligonucleotide. The structure of GalNAc₂-31_(a) is shownbelow:

The synthesis of an oligonucleotide comprising a GalNAc₂-32 conjugate isshown below.

Oligonucleotide 252 comprising a GalNAc₂-32 conjugate group, wherein Yis selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl,amino, substituted amino, azido, alkenyl or alkynyl, is synthesized asshown above. The GalNAc₂ cluster portion (GalNAc₂-32_(a)) of theconjugate group GalNAc₂-32 can be combined with any cleavable moiety toprovide a variety of conjugate groups. In certain embodiments, theY-containing group directly adjacent to the 5′-end of theoligonucleotide is part of the cleavable moiety. In certain embodiments,the Y-containing group directly adjacent to the 5′-end of theoligonucleotide is part of a stable moiety, and the cleavable moiety ispresent on the oligonucleotide. The structure of GalNAc₂-32_(a) is shownbelow:

Example 112: Modified Oligonucleotides Comprising a GalNAc₁ Conjugate

The oligonucleotides in Table 120 targeting SRB-1 were synthesized witha GalNAc₁ conjugate group in order to further test the potency ofoligonucleotides comprising conjugate groups that contain one GalNAcligand.

TABLE 120 GalNAc SEQ ISIS No. Sequence (5′ to 3′) cluster CM ID NO.711461 GalNAc ₁-25 _(a-o′) A _(do) G_(es) ^(m)C_(es) T_(es) T_(es)^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) GalNAc₁-25_(a) A_(d)145 T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es) T_(e) 711462 GalNAc ₁ -25 _(a-o′)G_(es) ^(m)C_(es) T_(es) T_(es)^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-25_(a)PO 143 G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es) T_(e) 711463 GalNAc ₁ -25 _(a-o′)G_(es) ^(m)C_(eo) T_(eo) T_(eo)^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-25_(a)PO 143 G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es) T_(e) 711465 GalNAc ₁ -26 _(a-o′) A _(do) G_(es) ^(m)C_(es)T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds)GalNAc₁-26_(a) A_(d) 145 T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es)^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711466 GalNAc ₁-26 _(a-o′)G_(es)^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds)A_(ds) T_(ds) GalNAc₁-26_(a) PO 143 G_(ds) A_(ds) ^(m)C_(ds) T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711467 GalNAc ₁ -26_(a-o′)G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds)^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-26_(a) PO 143 G_(ds) A_(ds) ^(m)C_(ds)T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(e) 711468 GalNAc ₁ -28_(a-o′) A _(do) G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds)T_(ds) ^(m)C_(ds) A_(ds) GalNAc₁-28_(a) A_(d) 145 T_(ds) G_(ds) A_(ds)^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711469GalNAc ₁ -28 _(a-o′)G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds)G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-28_(a) PO 143 G_(ds)A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e)711470 GalNAc ₁ -28 _(a-o′)G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo)A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-28_(a) PO 143G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es)T_(e) 713844 G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds)T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds)GalNAc₁-27_(a) PO 143 T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(eo′)-GalNAc ₁-27 _(a) 713845 G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo)A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds)T_(ds) GalNAc₁-27_(a) PO 143 T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(eo′)-GalNAc ₁-27 _(a) 713846 G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo)A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds)T_(ds) GalNAc₁-27_(a) A_(d) 144 T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es)T_(eo) A _(do′-) GalNAc ₁ -27 _(a) 713847 G_(es) ^(m)C_(es) T_(es)T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds)A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-29_(a) PO 143 T_(es) ^(m)C_(es)^(m)C_(es) T_(es) T_(eo′-) GalNAc ₁ -29 _(a) 713848 G_(es) ^(m)C_(eo)T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds)G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-29_(a) PO 143 T_(eo) ^(m)C_(eo)^(m)C_(es) T_(es) T_(eo′-) GalNAc ₁ -29 _(a) 713849 G_(es) ^(m)C_(es)T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds)G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-29_(a) A_(d) 144 T_(es)^(m)C_(es) ^(m)C_(es) T_(es) T_(eo) A _(do′-) GalNAc ₁ -29 _(a) 713850G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds)^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-29_(a)A_(d) 144 T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(eo) A _(do′-) GalNAc₁-29 _(a)

Example 113: Dose-Dependent Antisense Inhibition of Human Apolipoprotein(a) (Apo(a)) in Human Primary Hepatocytes

Selected gapmer antisense oligonucleotides from a previous publication(WO2005/000201, the content of which is incorporated by reference in itsentirety herein) were tested in a single dose assay in human primaryhepatocytes. Cells were obtained from Tissue Transformation Technologies(BD Biosciences, Franklin Lakes, N.J.) and treated with 150 nM ofantisense oligonucleotide. After a treatment period of approximately 16hours, RNA was isolated from the cells and apo(a) mRNA levels weremeasured by quantitative real-time PCR. Human apo(a) primer probe sethAPO(a)3′ (forward sequence ACAGCAATCAAACGAAGACACTG, designated hereinas SEQ ID NO: 5; reverse sequence AGCTTATACACAAAAATACCAAAAATGC,designated herein as SEQ ID NO: 6; probe sequenceTCCCAGCTACCAGCTATGCCAAACCTT, designated herein as SEQ ID NO: 7) was usedto measure mRNA levels. Additionally, mRNA levels were also measuredusing human apo(a) primer probe set hAPO(a)12kB (forward sequenceCCACAGTGGCCCCGGT, designated herein as SEQ ID NO: 8; reverse sequenceACAGGGCTTTTCTCAGGTGGT, designated herein as SEQ ID NO: 9; probe sequenceCCAAGCACAGAGGCTCCTTCTGAACAAG, designated herein as SEQ ID NO: 10).Apo(a) mRNA levels were normalized to GAPDH mRNA expression. Results arepresented in the table below as percent inhibition of apo(a), relativeto untreated control cells.

TABLE 121 Antisense inhibition of human apo(a) in human primaryhepatocytes % inhibition % inhibition ISIS (hAPO(a)3′ (hAPO(a)12 kB NoPPset) PPset) 144367 68 77 144368 42 59 144369 43 69 144370 80 75 14437142 57 144372 87 54 144373 63 49 144374 45 80 144375 33 11 144376 62 82144377 42 72 144378 0 72 144379 73 46 144380 75 78 144381 63 64 144382 058 144383 63 79 144384 38 0 144385 40 94 144386 47 61 144387 38 60144388 0 57 144389 52 39 144390 12 0 144391 73 57 144392 43 50 144393 8382 144394 40 76 144395 80 84 144396 53 72 144397 23 64 144398 7 33144399 43 44 144400 70 75 144401 87 72

Several antisense oligonucleotides were selected for further testing ina dose response assay.

The selected antisense oligonucleotides were tested in human primaryhepatocytes with 25 nM, 50 nM, 150 nM, or 300 nM concentrations ofantisense oligonucleotide, as specified in the table below. After atreatment period of approximately 16 hours, RNA was isolated from thecells and apo(a) mRNA levels were measured by quantitative real-timePCR. Human apo(a) primer probe set hAPO(a)3′ was used to measure mRNAlevels. Apo(a) mRNA levels were normalized to GAPDH mRNA expression.Results are presented as percent inhibition of apo(a), relative tountreated control cells.

TABLE 122 Dose-dependent antisense inhibition of human apo(a) in humanprimary hepatocytes, as measured with hAPO(a)3′ ISIS No 25 nM 50 nM 150nM 300 nM 144367 52 78 76 74 144370 64 74 68 66 144385 0 15 43 5 1443930 9 39 25 144395 17 9 8 32

ISIS 144367 demonstrated better efficacy and dose-dependency than theother antisense oligonucleotides. Hence, ISIS 144367 was considered thebenchmark antisense oligonucleotide to compare the potency of newlydesigned antisense oligonucleotides disclosed herein.

Example 114: Antisense Inhibition of Human Apo(a) in Transgenic MousePrimary Hepatocytes

Antisense oligonucleotides were newly designed targeting an apo(a)nucleic acid and were tested for their effects on apo(a) mRNA in vitro.The antisense oligonucleotides were tested for potency in a series ofparallel experiments that had similar culture conditions. Primaryhepatocytes from human apo(a) transgenic mice (Frazer, K. A. et al.,Nat. Genet. 1995. 9: 424-431) were used in this study. Hepatocytes at adensity of 35,000 cells per well were transfected using electroporationwith 1,000 nM antisense oligonucleotide. After a treatment period ofapproximately 24 hours, RNA was isolated from the cells and apo(a) mRNAlevels were measured by quantitative real-time PCR. Human primer probeset hAPO(a)12kB was used to measure mRNA levels. Apo(a) mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN®. Theresults for each experiment are presented in separate tables shownbelow. ISIS 144367 from was used as a benchmark for the new antisenseoligonucleotides and also included in the studies. Results are presentedas percent inhibition of apo(a), relative to untreated control cells. Atotal of 1,511 gapmers were tested under these culture conditions. Onlythose antisense oligonucleotides that were selected for further studyare presented in the table below with each table representing a separateexperiment.

The newly designed chimeric antisense oligonucleotides were designed as5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, whereinthe central gap segment comprises of ten 2′-deoxynucleosides and isflanked by wing segments on the 5′ direction and the 3′ directioncomprising five nucleosides each. Each nucleoside in the 5′ wing segmentand each nucleoside in the 3′ wing segment has a 2′-MOE modification.The internucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine residues throughout each gapmer are5-methylcytosines.

The apo(a) target sequence contains multiple Kringle repeat sequences,therefore, an antisense oligonucleotide may target one or more regionsof apo(a) depending whether on the oligonucleotide targets a Kringlesequence or not. “Start site” indicates the 5′-most nucleoside to whichthe gapmer is targeted in the human sequence. “Stop site” indicates the3′-most nucleoside to which the gapmer is targeted human sequence. Anapo(a) antisense oligonucleotide may have more than one “Start site” or“Stop site” depending on whether or not it targets a Kringle repeat.

Most gapmers listed in the tables are targeted with 100% complementarityto one or more regions of either the human apo(a) mRNA, designatedherein as SEQ ID NO: 1 (GENBANK Accession No. NM_005577.2) or the humanapo(a) genomic sequence, designated herein as SEQ ID NO: 2 (GENBANKAccession No. NT_007422.12 truncated from nucleotides 3230000 to3380000), or both. ‘n/a’ indicates that the antisense oligonucleotidedoes not target that particular sequence with 100% complementarity.

TABLE 123 SEQ ID SEQ SEQ NO: 1 SEQ ID ID NO: ID NO: SEQ ISIS Start NO: 1% 2 Start 2 Stop ID NO Site Stop Site Sequence inhibition Site Site NO144367 249 268 GGCAGGTCCTTCCTGTGACA 90 21210 21229 11 494157 238 257CCTGTGACAGTGGTGGAGTA 95 21199 21218 12 580 599 26690 26709 922 941 3223732256 1606 1625 43330 43349 1948 1967 48874 48893 2290 2309 54420 544393316 3335 72037 72056 494158 239 258 TCCTGTGACAGTGGTGGAGT 95 21200 2121913 581 600 26691 26710 923 942 32238 32257 1607 1626 43331 43350 19491968 48875 48894 2291 2310 54421 54440 3317 3336 72038 72057 494159 241260 CTTCCTGTGACAGTGGTGGA 97 21202 21221 14 583 602 26693 26712 925 94432240 32259 1609 1628 43333 43352 1951 1970 48877 48896 2293 2312 5442354442 3319 3338 72040 72059 4663 4682 94404 94423 5005 5024 115515115534 494160 242 261 CCTTCCTGTGACAGTGGTGG 97 21203 21222 15 4664 468394405 94424 5006 5025 115516 115535 494161 243 262 TCCTTCCTGTGACAGTGGTG96 21204 21223 16 4665 4684 94406 94425 5007 5026 115517 115536 494162244 263 GTCCTTCCTGTGACAGTGGT 95 21205 21224 17 3664 3683 77585 776044666 4685 94407 94426 5008 5027 115518 115537 494163 245 264GGTCCTTCCTGTGACAGTGG 96 21206 21225 18 4667 4686 94408 94427 494164 246265 AGGTCCTTCCTGTGACAGTG 93 21207 21226 19 4668 4687 94409 94428 494165247 266 CAGGTCCTTCCTGTGACAGT 91 21208 21227 20 4669 4688 94410 94429494166 248 267 GCAGGTCCTTCCTGTGACAG 89 21209 21228 21 494167 250 269TGGCAGGTCCTTCCTGTGAC 92 21211 21230 22 494168 251 270TTGGCAGGTCCTTCCTGTGA 89 21212 21231 23 494169 252 271CTTGGCAGGTCCTTCCTGTG 92 21213 21232 24 494170 253 272GCTTGGCAGGTCCTTCCTGT 88 21214 21233 25

TABLE 124 SEQ ID SEQ NO: 1 ID NO: SEQ ID SEQ ID Start 1 Stop % NO: 2 NO:2 SEQ ISIS NO Site Site Sequence inhibition Start Site Stop Site ID NO144367 249 268 GGCAGGTCCTTCCTGTGACA 91 21210 21229 11 84 494283 584 603TCTTCCTGTGACAGTGGTGG 93 26694 26713 26 926 945 32241 32260 1610 162943334 43353 1952 1971 48878 48897 2294 2313 54424 54443 3320 3339 7204172060 494284 585 604 TTCTTCCTGTGACAGTGGTG 95 26695 26714 27 927 94632242 32261 1611 1630 43335 43354 1953 1972 48879 48898 2295 2314 5442554444 3321 3340 72042 72061 494285 586 605 GTTCTTCCTGTGACAGTGGT 95 2669626715 28 928 947 32243 32262 1612 1631 43336 43355 1954 1973 48880 488992296 2315 54426 54445 3322 3341 72043 72062 494286 587 606GGTTCTTCCTGTGACAGTGG 95 26697 26716 29 929 948 32244 32263 1613 163243337 43356 1955 1974 48881 48900 2297 2316 54427 54446 494287 588 607AGGTTCTTCCTGTGACAGTG 95 26698 26717 30 930 949 32245 32264 1614 163343338 43357 1956 1975 48882 48901 2298 2317 54428 54447 494288 589 608CAGGTTCTTCCTGTGACAGT 91 26699 26718 31 931 950 32246 32265 1615 163443339 43358 1957 1976 48883 48902 2299 2318 54429 54448 2983 3002 6650066519 494290 592 611 TGGCAGGTTCTTCCTGTGAC 90 26702 26721 32 934 95332249 32268 1618 1637 43342 43361 1960 1979 48886 48905 2302 2321 5443254451 2986 3005 66503 66522 494291 593 612 TTGGCAGGTTCTTCCTGTGA 89 2670326722 33 935 954 32250 32269 1619 1638 43343 43362 1961 1980 48887 489062303 2322 54433 54452 2987 3006 66504 66523 494292 594 613CTTGGCAGGTTCTTCCTGTG 94 26704 26723 35 936 955 32251 32270 1620 163943344 43363 1962 1981 48888 48907 2304 2323 54434 54453 2988 3007 6650566524 494294 596 615 AGCTTGGCAGGTTCTTCCTG 90 26706 26725 36 938 95732253 32272 1622 1641 43346 43365 1964 1983 48890 48909 2306 2325 5443654455 2990 3009 66507 66526 494299 626 645 ACTATGCGAGTGTGGTGTCA 91 2673626755 37 968 987 32283 32302 1310 1329 37830 37849 1652 1671 43376 433951994 2013 48920 48939 2336 2355 54466 54485 2678 2697 60021 60040 30203039 66537 66556 494300 627 646 GACTATGCGAGTGTGGTGTC 93 26737 26756 38969 988 32284 32303 1311 1330 37831 37850 1653 1672 43377 43396 19952014 48921 48940 2337 2356 54467 54486 2679 2698 60022 60041 3021 304066538 66557 494301 628 647 CGACTATGCGAGTGTGGTGT 93 26738 26757 39 970989 32285 32304 1312 1331 37832 37851 1654 1673 43378 43397 1996 201548922 48941 2338 2357 54468 54487 2680 2699 60023 60042 3022 3041 6653966558 494302 629 648 CCGACTATGCGAGTGTGGTG 94 26739 26758 40 971 99032286 32305 1313 1332 37833 37852 1655 1674 43379 43398 1997 2016 4892348942 2339 2358 54469 54488 2681 2700 60024 60043 3023 3042 66540 66559494303 630 649 TCCGACTATGCGAGTGTGGT 93 26740 26759 41 972 991 3228732306 1314 1333 37834 37853 1656 1675 43380 43399 1998 2017 48924 489432340 2359 54470 54489 2682 2701 60025 60044 3024 3043 66541 66560 494304631 650 GTCCGACTATGCGAGTGTGG 94 26741 26760 42 973 992 32288 32307 13151334 37835 37854 1657 1676 43381 43400 1999 2018 48925 48944 2341 236054471 54490 2683 2702 60026 60045 3025 3044 66542 66561 494305 632 651GGTCCGACTATGCGAGTGTG 93 26742 26761 43 974 993 32289 32308 1316 133537836 37855 1658 1677 43382 43401 2000 2019 48926 48945 2342 2361 5447254491 2684 2703 60027 60046 3026 3045 66543 66562 494306 633 652GGGTCCGACTATGCGAGTGT 92 26743 26762 44 975 994 32290 32309 1317 133637837 37856 1659 1678 43383 43402 2001 2020 48927 48946 2343 2362 5447354492 2685 2704 60028 60047 3027 3046 66544 66563 494307 1190 1209CTGCTCAGTCGGTGCTTGTT 91 n/a n/a 45 2558 2577 494310 1193 1212CCTCTGCTCAGTCGGTGCTT 90 n/a n/a 46 2561 2580 494311 1194 1213GCCTCTGCTCAGTCGGTGCT 88 37714 37733 47 2562 2581 59905 59924 494334 12671286 CTTCCAGTGACAGTGGTGGA 90 37787 37806 48 2635 2654 59978 59997 4943361269 1288 TTCTTCCAGTGACAGTGGTG 90 37789 37808 49 2637 2656 59980 59999494337 1270 1289 GTTCTTCCAGTGACAGTGGT 95 37790 37809 50 2638 2657 5998160000 494338 1271 1290 GGTTCTTCCAGTGACAGTGG 91 37791 37810 133 2639 265859982 60001 494521 6393 6412 GACCTTAAAAGCTTATACAC 82 140049 140068 51494525 6397 6416 GTCAGACCTTAAAAGCTTAT 84 140053 140072 52 494530 64026421 TGTCAGTCAGACCTTAAAAG 82 140058 140077 53 494535 6407 6426GAATTTGTCAGTCAGACCTT 85 140063 140082 54 494536 6408 6427AGAATTTGTCAGTCAGACCT 83 140064 140083 55 494544 6417 6436CCTTAATACAGAATTTGTCA 82 140073 140092 56

TABLE 125 SEQ ID SEQ SEQ ID SEQ ID NO: 2 ID NO: ISIS NO: 1 NO: 1 % Start2 Stop SEQ NO Start Site Stop Site Sequence inhibition Site Site ID NO144367 249 268 GGCAGGTCCTTCCTGTGACA 84 21210 21229 11 494371 3900 3919GCTCCGTTGGTGCTTGTTCA 93 n/a n/a 57 494372 3901 3920 TGCTCCGTTGGTGCTTGTTC93 n/a n/a 58 494373 3902 3921 TTGCTCCGTTGGTGCTTGTT 83 n/a n/a 59 4943743903 3922 TTTGCTCCGTTGGTGCTTGT 89 n/a n/a 60 494375 3904 3923CTTTGCTCCGTTGGTGCTTG 85 n/a n/a 61 494386 3977 3996 TCCTGTAACAGTGGTGGAGA86 81985 82004 62 494387 3978 3997 TTCCTGTAACAGTGGTGGAG 82 81986 8200563 494388 3979 3998 CTTCCTGTAACAGTGGTGGA 86 81987 82006 64 494389 39803999 CCTTCCTGTAACAGTGGTGG 92 81988 82007 65 494390 3981 4000TCCTTCCTGTAACAGTGGTG 92 81989 82008 66 494391 3982 4001GTCCTTCCTGTAACAGTGGT 84 81990 82009 67 494392 3983 4002TGTCCTTCCTGTAACAGTGG 81 81991 82010 68

TABLE 126 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISISNO Start Site Stop Site Sequence inhibition Start Site Stop Site ID NO144367 249 268 GGCAGGTCCTTCCTGTGACA 86 21210 21229 11 498369 3203 3222TGGAGCCAGAATAACATTCG 91 70667 70686 69 498379 3213 3232CCTCTAGGCTTGGAGCCAGA 85 70677 70696 70 498408 3323 3342AGTTCTTCCTGTGACAGTGG 86 72044 72063 71 498433 3367 3386GTCCGACTATGCTGGTGTGG 87 72088 72107 72 498434 3368 3387GGTCCGACTATGCTGGTGTG 86 72089 72108 73 498435 3369 3388GGGTCCGACTATGCTGGTGT 83 72090 72109 74

TABLE 127 SEQ ID SEQ ID SEQ ID SEQ ID ISIS NO: 1 NO: 1 % NO: 2 NO: 2 SEQNO Start Site Stop Site Sequence inhibition Start Site Stop Site ID NO144367 249 268 GGCAGGTCCTTCCTGTGACA 90 21210 21229 11 498229 2871 2890CCTCTAGGCTTGGAATCGGG 90 65117 65136 75 498238 2883 2902GTTCAGAAGGAGCCTCTAGG 93 65129 65148 76 498239 2884 2903TGTTCAGAAGGAGCCTCTAG 94 65130 65149 77 498240 2887 2906GCTTGTTCAGAAGGAGCCTC 98 n/a n/a 78 4573 4592 498241 2888 2907TGCTTGTTCAGAAGGAGCCT 94 n/a n/a 79 4574 4593 498242 2889 2908GTGCTTGTTCAGAAGGAGCC 96 n/a n/a 80 4575 4594 498243 2890 2909GGTGCTTGTTCAGAAGGAGC 97 n/a n/a 81 4576 4595 498244 2891 2910TGGTGCTTGTTCAGAAGGAG 92 n/a n/a 82 4577 4596 498251 2898 2917GCTCAGTTGGTGCTTGTTCA 90 n/a n/a 83 498252 2899 2918 TGCTCAGTTGGTGCTTGTTC90 n/a n/a 84

TABLE 128 SEQ ID SEQ ID SEQ ID SEQ ID SEQ NO: 1 NO: 1 % NO: 2 NO: 2 IDISIS NO Start Site Stop Site Sequence inhibition Start Site Stop Site NO144367 249 268 GGCAGGTCCTTCCTGTGACA 91 21210 21229 11 498517 3548 3567GCTTGGATCTGGGACCACCG 89 76233 76252 85

TABLE 129 SEQ ID SEQ ID NO: 1 NO: 1 SEQ ID SEQ ID SEQ ISIS Start Stop %NO: 2 NO: 2 ID NO Site Site Sequence inhibition Start Site Stop Site NO144367 249 268 GGCAGGTCCTTCCTGTGACA 94  21210  21229 11 498833 4900 4919GCCTCCATGCTTGGAACTGG 94 114205 114224 86 498859 4926 4945GCTCAGTTGGTGCTGCTTCA 92 n/a n/a 87 498868 4978 4997 CCTCGATAACTCTGGCCATT94 115488 115507 88 498875 5003 5022 TCCTGTGACAGTGGTGGAGA 94 115513115532 89

TABLE 130 SEQ ID SEQ ID NO: 1 NO: 1 SEQ ID SEQ ID ISIS Start Stop % NO:2 NO: 2 SEQ NO Site Site Sequence inhibition Start Site Stop Site ID NO144367 249 268 GGCAGGTCCTTCCTGTGACA 92 21210 21229 11 499020 6257 6276GTAGGTTGATGCTTCACTCT 91 139913 139932 90 499041 6318 6337CGTTTGATTGCTGTCTATTA 90 139974 139993 91

TABLE 131 SEQ ID SEQ ID SEQ ID SEQ ID ISIS NO: 1 NO: 1 % NO: 2 NO: 2 SEQNO Start Site Stop Site Sequence inhibition Start Site Stop Site ID NO144367 249 268 GGCAGGTCCTTCCTGTGACA 91 21210 21229 11 498523 3554 3573CTCTGTGCTTGGATCTGGGA 94 76239 76258 92 498524 3555 3574CCTCTGTGCTTGGATCTGGG 96 76240 76259 93 498525 3556 3575GCCTCTGTGCTTGGATCTGG 94 76241 76260 94 498529 3560 3579AGAAGCCTCTGTGCTTGGAT 89 76245 76264 95 498535 3566 3585TTCAGAAGAAGCCTCTGTGC 89 76251 76270 96 498550 3582 3601GCTCCGTTGGTGCTTCTTCA 90 n/a n/a 97 498553 3585 3604 TTTGCTCCGTTGGTGCTTCT87 n/a n/a 98 498555 3587 3606 GCTTTGCTCCGTTGGTGCTT 90 n/a n/a 99 39053924 498556 3588 3607 GGCTTTGCTCCGTTGGTGCT 89 77509 77528 100 3906 392581914 81933 498557 3589 3608 GGGCTTTGCTCCGTTGGTGC 89 77510 77529 1013907 3926 81915 81934 498579 3662 3681 CCTTCCTGTGACAGTGGTAG 87 7758377602 102 498580 3663 3682 TCCTTCCTGTGACAGTGGTA 92 77584 77603 103498581 3665 3684 TGTCCTTCCTGTGACAGTGG 94 77586 77605 104 5009 5028115519 115538

TABLE 132 SEQ ID SEQ ID SEQ ID SEQ ID ISIS NO: 1 NO: 1 % NO: 2 NO: 2 SEQID NO Start Site Stop Site Sequence inhibition Start Site Stop Site NO144367 249 268 GGCAGGTCCTTCCTGTGACA 100 21210 21229 11 494230 477 496CCTCTAGGCTTGGAACCGGG 95 25380 25399 105 819 838 30927 30946 1161 118036471 36490 1503 1522 42020 42039 1845 1864 47564 47583 2187 2206 5311053129 2529 2548 58662 58681 494243 494 513 TGCTTGTTCGGAAGGAGCCT 93 n/an/a 106 836 855 1178 1197 1520 1539 1862 1881 2204 2223 2546 2565 494244495 514 GTGCTTGTTCGGAAGGAGCC 95 n/a n/a 107 837 856 1179 1198 1521 15401863 1882 2205 2224 2547 2566

TABLE 133 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 % NO: 2 NO: 2 SEQ ISISNO Start Site Stop Site Sequence inhibition Start Site Stop Site ID NO144367 249 268 GGCAGGTCCTTCCTGTGACA 96 21210 21229 11 494466 4208 4227GCTTGGAACTGGGACCACCG 95 85138 85157 108 494470 4212 4231CTGTGCTTGGAACTGGGACC 94 85142 85161 109 494472 4214 4233CTCTGTGCTTGGAACTGGGA 92 85144 85163 110

Example 115: Dose-Dependent Antisense Inhibition of Apo(a) in TransgenicMouse Primary Hepatocytes

Gapmers from the studies described above exhibiting significant in vitroinhibition of apo(a) mRNA were selected and tested at various doses intransgenic mouse primary hepatocytes in a series of parallel studieswith similar culture conditions. Cells were plated at a density of35,000 per well and transfected using electroporation with 0.0625 μM,0.125 μM, 0.25 μM, 0.500 μM, or 1.000 μM concentrations of antisenseoligonucleotide. After a treatment period of approximately 16 hours, RNAwas isolated from the cells and apo(a) mRNA levels were measured byquantitative real-time PCR. Apo(a) primer probe set hAPO(a)12kB was usedto measured mRNA levels. Apo(a) mRNA levels were adjusted according tototal RNA content, as measured by RIBOGREEN®. Results are presented aspercent inhibition of apo(a), relative to untreated control cells.

The results of each of the studies are depicted in the tables presentedbelow with each table representing a separate experiment. The halfmaximal inhibitory concentration (IC₅₀) of each oligonucleotide is alsopresented in the tables. Apo(a) mRNA levels were significantly reducedin a dose-dependent manner in antisense oligonucleotide treated cells.The potency of the newly designed oligos was compared with the benchmarkoligonucleotide ISIS 144367.

TABLE 134 0.0625 0.125 0.250 0.500 1.000 IC₅₀ ISIS No μM μM μM μM μM(μM) 144367 11 27 46 62 80 0.31 494157 11 47 53 76 87 0.23 494158 19 5775 84 88 0.13 494159 41 65 77 84 92 0.07 494160 44 69 76 85 91 0.06494161 40 64 74 85 91 0.08 494162 36 63 76 87 88 0.09 494163 20 59 75 8592 0.13 494164 3 45 62 74 90 0.21 494165 25 39 57 71 75 0.19 494166 1730 47 59 76 0.31 494167 30 43 55 72 80 0.18 494168 25 36 44 59 75 0.28494169 19 39 51 61 81 0.25

TABLE 135 0.0625 0.125 0.250 0.500 1.000 IC₅₀ ISIS No μM μM μM μM μM(μM) 144367 23 40 58 76 88 0.19 494170 38 34 60 76 84 0.13 494230 55 7189 95 97 0.03 494243 47 73 87 92 97 0.05 494244 58 73 86 92 96 0.03494283 54 70 84 93 94 0.05 494284 45 62 83 92 95 0.07 494285 56 70 84 9295 0.04 494286 51 70 87 93 95 0.05 494287 32 60 67 87 91 0.11 494288 2641 61 79 88 0.17 494290 30 43 64 81 87 0.15 494291 29 40 56 75 85 0.18

TABLE 136 0.0625 0.125 0.250 0.500 1.000 IC₅₀ ISIS No μM μM μM μM μM(μM) 144367 10 38 62 68 84 0.23 494292 17 36 74 85 90 0.17 494294 10 3453 80 91 0.22 494299 32 29 56 77 88 0.16 494300 34 46 76 86 90 0.12494301 44 56 72 86 89 0.09 494302 42 59 78 88 89 0.08 494303 37 58 70 8689 0.10 494304 46 71 78 89 90 0.05 494305 39 58 62 85 87 0.10 494306 3152 65 79 88 0.13 494307 23 23 39 65 78 0.34 494310 14 29 62 70 88 0.25

TABLE 137 0.0625 0.125 0.250 0.500 1.000 IC₅₀ ISIS No μM μM μM μM μM(μM) 144367 0 29 45 73 92 0.27 494311 28 53 65 85 95 0.13 494334 20 4466 86 96 0.16 494336 15 38 54 84 97 0.20 494337 28 50 77 90 98 0.12494338 21 40 68 91 98 0.15 494371 19 0 71 89 97 0.15 494372 33 44 77 9197 0.12 494373 15 36 65 83 95 0.19 494374 3 17 51 83 90 0.24 494375 1 3456 80 93 0.23 494386 13 26 46 73 91 0.25 494387 17 27 45 67 88 0.28

TABLE 138 0.0625 0.125 0.250 0.500 1.000 IC₅₀ ISIS No μM μM μM μM μM(μM) 144367 35 42 62 70 91 0.15 494537 19 34 54 79 90 0.21 494544 10 3873 86 94 0.17 498229 36 58 80 92 97 0.10 498238 41 57 75 91 97 0.09498239 56 71 79 90 94 0.03 498240 91 94 98 99 100 <0.06 498241 75 84 9196 98 <0.06 498242 11 27 42 47 63 0.49 498243 91 93 96 98 99 <0.06498244 4 0 0 13 43 >1.00 498251 30 30 42 73 89 0.26 498252 37 33 58 8092 0.20 498369 22 22 10 22 34 >1.00

TABLE 139 0.0625 0.125 0.250 0.500 1.000 IC₅₀ ISIS No μM μM μM μM μM(μM) 144367 15 32 54 75 90 0.22 498379 29 48 71 80 95 0.13 498408 38 5777 88 96 0.09 498433 29 36 70 88 96 0.15 498434 49 43 50 78 90 0.19498435 27 39 57 78 93 0.18 498517 64 72 82 93 98 <0.06 498721 77 84 8896 97 <0.06 498833 73 78 91 95 99 <0.06 498859 7 24 37 62 75 0.36 4988687 14 39 63 81 0.36 498875 16 21 33 55 81 0.39 499020 7 24 23 55 78 0.36499041 6 16 33 64 83 0.35

TABLE 140 0.0625 0.125 0.250 0.500 1.000 IC₅₀ ISIS No μM μM μM μM μM(μM) 144367 14 47 64 79 91 0.14 498523 36 50 80 87 95 0.11 498524 43 7987 93 97 0.01 498525 32 49 75 86 96 0.12 498529 21 49 57 78 90 0.17498535 20 34 55 76 86 0.21 498550 12 50 69 84 96 0.11 498553 8 43 55 7791 0.21 498555 13 35 68 86 94 0.19 498556 27 37 71 85 91 0.15 498557 1842 75 89 95 0.16 498579 16 38 67 89 95 0.16 498580 36 57 81 91 96 0.10498581 34 64 75 93 97 0.05

TABLE 141 0.0625 0.125 0.250 0.500 1.000 IC₅₀ ISIS No μM μM μM μM μM(μM) 144367 0 9 26 49 77 0.47 494388 0 0 21 33 55 0.89 494389 0 15 22 5079 0.46 494390 5 20 37 68 81 0.33 494391 7 20 32 54 68 0.46 494392 18 2440 57 76 0.35 494466 33 45 58 69 82 0.16 494470 45 58 68 79 87 0.08494472 37 50 60 69 83 0.13 494521 0 0 0 15 54 0.17 494525 0 0 2 28 650.85 494530 0 6 27 51 80 0.46 494535 0 7 24 53 74 0.49 494536 0 2 15 4267 0.63

TABLE 142 0.0625 0.125 0.250 0.500 1.000 IC₅₀ ISIS No μM μM μM μM μM(μM) 144367 0 4 16 26 77 0.65 498379 12 18 27 32 63 0.81 498408 0 11 4650 77 0.41 498433 22 30 46 60 83 0.27 498434 39 29 25 47 78 0.40 49843521 28 26 43 73 0.50 498517 44 48 63 70 84 0.11 498721 54 54 66 75 89<0.06 498833 44 51 58 67 83 0.11 498859 0 29 14 35 66 0.69 498868 0 12 926 60 1.07 498875 0 30 31 53 78 0.40 499020 0 27 19 45 74 0.51 499041 012 10 37 65 0.77As presented in the tables above, ISIS 494157 (SEQ ID NO: 12), ISIS494158 (SEQ ID NO:13), ISIS 494159 (SEQ ID NO:14), ISIS 494160 (SEQ IDNO: 15), ISIS 494161 (SEQ ID NO:16), ISIS 494162 (SEQ ID NO: 17), ISIS494163 (SEQ ID NO: 18), ISIS 494164 (SEQ ID NO: 19), ISIS 494165 (SEQ IDNO: 20), ISIS 494167 (SEQ ID NO: 22), ISIS 494168 (SEQ ID NO: 23), ISIS494169 (SEQ ID NO: 24), ISIS 494170 (SEQ ID NO: 25), ISIS 494230 (SEQ IDNO: 105), ISIS 494243 (SEQ ID NO: 106), ISIS 494244 (SEQ ID NO: 107),ISIS 494283 (SEQ ID NO: 26), ISIS 494284 (SEQ ID NO: 27), ISIS 494285(SEQ ID NO: 28), ISIS 494286 (SEQ ID NO: 29), ISIS 494287 (SEQ ID NO:30), ISIS 494288 (SEQ ID NO: 31), ISIS 494290 (SEQ ID NO: 32), ISIS494291 (SEQ ID NO: 33), ISIS 494292 (SEQ ID NO: 35), ISIS 494294 (SEQ IDNO: 36), ISIS 494299 (SEQ ID NO: 37), ISIS 494300 (SEQ ID NO: 38), ISIS494301 (SEQ ID NO: 39), ISIS 494302 (SEQ ID NO: 40), ISIS 494303 (SEQ IDNO: 41), ISIS 494304 (SEQ ID NO: 42), ISIS 494305 (SEQ ID NO:43), ISIS494306 (SEQ ID NO: 44), ISIS 494311 (SEQ ID NO: 47), ISIS 494334 (SEQ IDNO: 48), ISIS 494336 (SEQ ID NO: 49), ISIS 494337 (SEQ ID NO: 50), ISIS494338 (SEQ ID NO: 133), ISIS 494371 (SEQ ID NO: 57), ISIS 494372 (SEQID NO: 58), ISIS 494373 (SEQ ID NO: 59), ISIS 494374 (SEQ ID NO: 60),ISIS 494375 (SEQ ID NO: 61), ISIS 494386 (SEQ ID NO: 62), ISIS 494389(SEQ ID NO: 65), ISIS 494390 (SEQ ID NO: 66), ISIS 494392 (SEQ ID NO:68), ISIS 494466 (SEQ ID NO: 108), ISIS 494470 (SEQ ID NO: 109), ISIS494472 (SEQ ID NO: 110), ISIS 494521 (SEQ ID NO: 51), ISIS 494530 (SEQID NO: 53), ISIS 498229 (SEQ ID NO: 75), ISIS 498238 (SEQ ID NO: 76),ISIS 498239 (SEQ ID NO: 77), ISIS 498240 (SEQ ID NO: 78), ISIS 498241(SEQ ID NO: 79), ISIS 498243 (SEQ ID NO: 81), ISIS 498379 (SEQ ID NO:70), ISIS 498408 (SEQ ID NO: 71), ISIS 498433 (SEQ ID NO: 72), ISIS498434 (SEQ ID NO: 73), ISIS 498435 (SEQ ID NO: 74), ISIS 498517 (SEQ IDNO: 85), ISIS 498523 (SEQ ID NO: 92), ISIS 498524 (SEQ ID NO: 93), ISIS498525 (SEQ ID NO: 94), ISIS 498550 (SEQ ID NO: 97), ISIS 498580 (SEQ IDNO: 103), ISIS 498581 (SEQ ID NO: 104), ISIS 498721(ATGCCTCGATAACTCCGTCC; SEQ ID NO: 134), ISIS 498833 (SEQ ID NO: 86),ISIS 498875 (SEQ ID NO: 89), and ISIS 499020 (SEQ ID NO: 90) were morepotent than ISIS 144367 (SEQ ID NO: 11).

Example 116: Dose-Dependent Antisense Inhibition of Apo(a) in TransgenicMouse Primary Hepatocytes

Potent gapmers from the studies described above were further selectedand tested at various doses in transgenic mouse primary hepatocytes in aseries of studies with similar culture conditions. Cells were plated ata density of 35,000 per well and transfected using electroporation with0.049 μM, 0.148 μM, 0.444 μM, 1.333 μM, or 4.000 μM concentrations ofantisense oligonucleotide, as specified in tables below. After atreatment period of approximately 16 hours, RNA was isolated from thecells and apo(a) mRNA levels were measured by quantitative real-timePCR. Apo(a) primer probe set hAPO(a)12kB was used to measured mRNAlevels. Apo(a) mRNA levels were adjusted according to total RNA content,as measured by RIBOGREEN®. Results are presented as percent inhibitionof apo(a), relative to untreated control cells.

The results of each of the studies are depicted in the tables presentedbelow with each table representing a separate experiment. The halfmaximal inhibitory concentration (IC₅₀) of each oligonucleotide is alsopresented in the tables. Apo(a) mRNA levels were significantly reducedin a dose-dependent manner in antisense oligonucleotide treated cells.The potency of the newly designed oligos was compared with the benchmarkoligonucleotide, ISIS 144367. As presented in the tables below, ISIS494157 (SEQ ID NO: 12), ISIS 494158 (SEQ ID NO:13), ISIS 494159 (SEQ IDNO:14), ISIS 494160 (SEQ ID NO: 15), ISIS 494161 (SEQ ID NO:16), ISIS494162 (SEQ ID NO: 17), ISIS 494163 (SEQ ID NO: 18), ISIS 494164 (SEQ IDNO: 19), ISIS 494230 (SEQ ID NO: 105), ISIS 494243 (SEQ ID NO: 106),ISIS 494244 (SEQ ID NO: 107), ISIS 494283 (SEQ ID NO: 26), ISIS 494284(SEQ ID NO: 27), ISIS 494285 (SEQ ID NO: 28), ISIS 494286 (SEQ ID NO:29), ISIS 494287 (SEQ ID NO: 30), ISIS 494290 (SEQ ID NO: 32), ISIS494292 (SEQ ID NO: 35), ISIS 494300 (SEQ ID NO: 38), ISIS 494301 (SEQ IDNO: 39), ISIS 494302 (SEQ ID NO: 40), ISIS 494303 (SEQ ID NO: 41), ISIS494304 (SEQ ID NO: 42), ISIS 494305 (SEQ ID NO: 43), ISIS 494306 (SEQ IDNO: 44), ISIS 494310 (SEQ ID NO: 46), ISIS 494311 (SEQ ID NO: 47), ISIS494337 (SEQ ID NO: 50), ISIS 494371 (SEQ ID NO: 57), ISIS 494372 (SEQ IDNO: 58), ISIS 494375 (SEQ ID NO: 61), ISIS 494388 (SEQ ID NO: 64), ISIS494389 (SEQ ID NO: 65), ISIS 494390 (SEQ ID NO: 66), ISIS 494392 (SEQ IDNO: 68), ISIS 494466 (SEQ ID NO: 108), ISIS 494470 (SEQ ID NO: 109),ISIS 494472 (SEQ ID NO: 110), ISIS 498238 (SEQ ID NO: 76), ISIS 498239(SEQ ID NO: 77), ISIS 498433 (SEQ ID NO: 72), ISIS 498434 (SEQ ID NO:73), ISIS 498435 (SEQ ID NO: 74), ISIS 498523 (SEQ ID NO: 92), ISIS498524 (SEQ ID NO: 93), ISIS 498525 (SEQ ID NO: 94), ISIS 498580 (SEQ IDNO: 103), and ISIS 498581 (SEQ ID NO: 104) were more potent than ISIS144367 (SEQ ID NO: 11).

TABLE 143 0.049 0.148 0.444 1.333 4.000 IC₅₀ ISIS No μM μM μM μM μM (μM)144367 0 26 67 89 92 0.32 494157 23 50 83 96 96 0.15 494158 26 62 85 9696 0.11 494159 42 65 87 95 94 0.07 494160 51 70 88 94 94 <0.05 494161 3667 87 95 96 0.08 494162 40 69 89 94 95 0.07 494163 41 57 87 95 94 0.08494164 15 43 75 93 96 0.20 494230 39 77 94 99 99 0.05 494243 39 76 92 9899 0.06 494244 58 79 91 97 99 0.02 494283 18 45 80 93 91 0.18 494284 953 80 95 94 0.18

TABLE 144 0.049 0.148 0.444 1.333 4.000 IC₅₀ ISIS No μM μM μM μM μM (μM)144367 21 40 79 94 93 0.18 494285 53 68 90 97 97 <0.05 494286 46 69 8996 97 0.05 494287 31 38 79 94 95 0.15 494290 22 53 74 93 94 0.16 49429237 51 81 93 95 0.11 494294 22 40 72 91 94 0.19 494299 15 43 75 93 950.20 494300 25 38 79 95 95 0.17 494301 23 48 82 92 95 0.15 494302 26 5986 93 94 0.12 494303 10 58 84 92 91 0.16 494304 25 62 83 93 93 0.12

TABLE 145 0.049 0.148 0.444 1.333 4.000 IC₅₀ ISIS No μM μM μM μM μM (μM)144367 23 40 70 90 94 0.19 494305 20 48 82 93 95 0.16 494306 26 53 78 9192 0.14 494310 36 50 79 88 92 0.12 494311 38 50 74 93 95 0.12 494334 2042 73 90 94 0.19 494336 5 39 74 92 95 0.23 494337 23 51 87 96 96 0.14494338 12 42 82 93 95 0.19 494371 28 49 82 94 94 0.14 494372 28 54 81 9388 0.13 494373 21 28 67 86 92 0.25 494375 26 40 77 85 92 0.18

TABLE 146 0.049 0.148 0.444 1.333 4.000 IC₅₀ ISIS No μM μM μM μM μM (μM)144367 5 33 65 78 81 0.32 494388 30 32 60 82 86 0.25 494389 30 45 69 8484 0.17 494390 32 47 67 83 87 0.16 494392 23 38 54 79 82 0.31 494466 4867 86 91 95 0.04 494470 74 87 92 96 98 <0.05 494472 69 84 92 96 97 <0.05494544 5 18 49 74 79 0.48 498238 25 51 76 92 96 0.15 498239 25 62 83 9397 0.12 498379 5 21 53 71 81 0.55 498408 1 38 63 79 80 0.32 498433 23 4370 77 79 0.21

TABLE 147 0.049 0.148 0.444 1.333 4.000 IC₅₀ ISIS No μM μM μM μM μM (μM)144367 0 40 76 90 93 0.26 498434 32 44 64 78 84 0.20 498435 24 42 64 7779 0.23 498517 28 23 53 81 85 0.45 498523 50 64 81 90 93 <0.05 498524 5370 84 93 96 <0.05 498525 38 55 80 92 96 0.09 498550 12 18 62 81 83 0.33498557 13 33 67 79 83 0.33 498579 6 42 69 80 85 0.31 498580 6 46 76 8283 0.23 498581 5 40 78 81 84 0.25 498721 40 31 58 78 83 0.35 498833 2120 58 80 90 0.44

Example 117: Antisense Inhibition of Human Apo(a) in Transgenic MousePrimary Hepatocytes

Additional antisense oligonucleotides were newly designed targeting anapo(a) nucleic acid and were tested for their effects on apo(a) mRNA invitro. The antisense oligonucleotides were tested in a series ofexperiments that had similar culture conditions. Primary hepatocytesfrom human apo(a) transgenic mice were used in this study. Hepatocytesat a density of 35,000 cells per well were transfected usingelectroporation with 1,000 nM antisense oligonucleotide. After atreatment period of approximately 24 hours, RNA was isolated from thecells and apo(a) mRNA levels were measured by quantitative real-timePCR. Human primer probe set hAPO(a)12kB was used to measure mRNA levels.Apo(a) mRNA levels were adjusted according to total RNA content, asmeasured by RIBOGREEN®. The results for each experiment are presented inseparate tables shown below. ISIS 144367 was also included in thestudies for comparison. Results are presented as percent inhibition ofapo(a), relative to untreated control cells. A total of 231 antisenseoligonucleotides were tested under these culture conditions. Only thoseantisense oligonucleotides that were selected for further studies arepresented below.

The newly designed chimeric antisense oligonucleotides were designed as3-10-4 MOE gapmers. The gapmers are 17 nucleosides in length, whereinthe central gap segment comprises of ten 2′-deoxynucleosides and isflanked by wing segments on the 5′ direction and the 3′ directioncomprising three nucleosides and four nucleosides respectively. Eachnucleoside in the 5′ wing segment and each nucleoside in the 3′ wingsegment has a 2′-MOE modification. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P═S) linkages. All cytosineresidues throughout each gapmer are 5-methylcytosines.

The apo(a) target sequence contains multiple Kringle repeat sequences,therefore, an antisense oligonucleotide may target one or more regionsof apo(a) depending whether on the oligonucleotide targets a Kringlesequence or not. “Start site” indicates the 5′-most nucleoside to whichthe gapmer is targeted in the human sequence. “Stop site” indicates the3′-most nucleoside to which the gapmer is targeted human sequence. Anapo(a) antisense oligonucleotide may have more than one “Start site” or“Stop site” depending on whether or not it targets a Kringle repeat.

Most gapmers listed in the tables are targeted with 100% complementarityto multiple regions of either the human apo(a) mRNA, designated hereinas SEQ ID NO: 1 (GENBANK Accession No. NM_005577.2) or the human apo(a)genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK AccessionNo. NT_007422.12 truncated from nucleotides 3230000 to 3380000), orboth. ‘n/a’ indicates that the antisense oligonucleotide does not targetthat particular sequence with 100% complementarity.

TABLE 148 SEQ ID SEQ ID SEQ ID SEQ ID ISIS NO: 1 NO: 1 % NO: 2 NO: 2 SEQID NO Start Site Stop Site Sequence inhibition Start Site Stop Site NO144367 249 268 GGCAGGTCCTTCCTGTGACA 64 21210 21229 11 510542 241 257CCTGTGACAGTGGTGGA 79 21202 21218 111 583 599 CCTGTGACAGTGGTGGA 2669326709 925 941 CCTGTGACAGTGGTGGA 32240 32256 1609 1625 CCTGTGACAGTGGTGGA43333 43349 1951 1967 CCTGTGACAGTGGTGGA 48877 48893 2293 2309CCTGTGACAGTGGTGGA 54423 54439 3319 3335 CCTGTGACAGTGGTGGA 72040 720564663 4679 CCTGTGACAGTGGTGGA 94404 94420 5005 5021 CCTGTGACAGTGGTGGA115515 115531 510543 242 258 TCCTGTGACAGTGGTGG 75 21203 21219 112 584600 TCCTGTGACAGTGGTGG 26694 26710 926 942 TCCTGTGACAGTGGTGG 32241 322571610 1626 TCCTGTGACAGTGGTGG 43334 43350 1952 1968 TCCTGTGACAGTGGTGG48878 48894 2294 2310 TCCTGTGACAGTGGTGG 54424 54440 3320 3336TCCTGTGACAGTGGTGG 72041 72057 4664 4680 TCCTGTGACAGTGGTGG 94405 944215006 5022 TCCTGTGACAGTGGTGG 115516 115532 510544 243 259TTCCTGTGACAGTGGTG 73 21204 21220 113 585 601 TTCCTGTGACAGTGGTG 2669526711 927 943 TTCCTGTGACAGTGGTG 32242 32258 1611 1627 TTCCTGTGACAGTGGTG43335 43351 1953 1969 TTCCTGTGACAGTGGTG 48879 48895 2295 2311TTCCTGTGACAGTGGTG 54425 54441 3321 3337 TTCCTGTGACAGTGGTG 72042 720584665 4681 TTCCTGTGACAGTGGTG 94406 94422 5007 5023 TTCCTGTGACAGTGGTG115517 115533 510545 244 260 CTTCCTGTGACAGTGGT 65 21205 21221 114 586602 CTTCCTGTGACAGTGGT 26696 26712 928 944 CTTCCTGTGACAGTGGT 32243 322591612 1628 CTTCCTGTGACAGTGGT 43336 43352 1954 1970 CTTCCTGTGACAGTGGT48880 48896 2296 2312 CTTCCTGTGACAGTGGT 54426 54442 3322 3338CTTCCTGTGACAGTGGT 72043 72059 3664 3680 CTTCCTGTGACAGTGGT 77585 776014666 4682 CTTCCTGTGACAGTGGT 94407 94423 5008 5024 CTTCCTGTGACAGTGGT115518 115534 510546 245 261 CCTTCCTGTGACAGTGG 74 21206 21222 115 36653681 CCTTCCTGTGACAGTGG 77586 77602 4667 4683 CCTTCCTGTGACAGTGG 9440894424 5009 5025 CCTTCCTGTGACAGTGG 115519 115535 510547 246 262TCCTTCCTGTGACAGTG 77 21207 21223 116 3666 3682 TCCTTCCTGTGACAGTG 7758777603 4668 4684 TCCTTCCTGTGACAGTG 94409 94425 5010 5026TCCTTCCTGTGACAGTG 115520 115536 510548 247 263 GTCCTTCCTGTGACAGT 7321208 21224 117 3667 3683 GTCCTTCCTGTGACAGT 77588 77604 4669 4685GTCCTTCCTGTGACAGT 94410 94426 5011 5027 GTCCTTCCTGTGACAGT 115521 115537510549 248 264 GGTCCTTCCTGTGACAG 67 21209 21225 118 4670 4686GGTCCTTCCTGTGACAG 94411 94427 510595 632 648 CCGACTATGCGAGTGTG 76 2674226758 119 974 990 CCGACTATGCGAGTGTG 32289 32305 1316 1332CCGACTATGCGAGTGTG 37836 37852 1658 1674 CCGACTATGCGAGTGTG 43382 433982000 2016 CCGACTATGCGAGTGTG 48926 48942 2342 2358 CCGACTATGCGAGTGTG54472 54488 2684 2700 CCGACTATGCGAGTGTG 60027 60043 3026 3042CCGACTATGCGAGTGTG 66543 66559 510597 634 650 GTCCGACTATGCGAGTG 70 2674426760 120 976 992 GTCCGACTATGCGAGTG 32291 32307 1318 1334GTCCGACTATGCGAGTG 37838 37854 1660 1676 GTCCGACTATGCGAGTG 43384 434002002 2018 GTCCGACTATGCGAGTG 48928 48944 2344 2360 GTCCGACTATGCGAGTG54474 54490 2686 2702 GTCCGACTATGCGAGTG 60029 60045 3028 3044GTCCGACTATGCGAGTG 66545 66561 510598 635 651 GGTCCGACTATGCGAGT 70 2674526761 121 977 993 GGTCCGACTATGCGAGT 32292 32308 1319 1335GGTCCGACTATGCGAGT 37839 37855 1661 1677 GGTCCGACTATGCGAGT 43385 434012003 2019 GGTCCGACTATGCGAGT 48929 48945 2345 2361 GGTCCGACTATGCGAGT54475 54491 2687 2703 GGTCCGACTATGCGAGT 60030 60046 3029 3045GGTCCGACTATGCGAGT 66546 66562

TABLE 149 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ISIS NO: 1 NO: 1 % NO: 2 NO: 2ID NO Start Site Stop Site Sequence inhibition Start Site Stop Site NO144367 249 268 GGCAGGTCCTTCCTGTGACA 83 21210 21229 11 510783 6400 6416GTCAGACCTTAAAAGCT 75 140056 140072 122 512944 3561 3577AAGCCTCTGTGCTTGGA 81 76246 76262 123 512947 3560 3576 AGCCTCTGTGCTTGGAT85 76245 76261 124 512958 3559 3575 GCCTCTGTGCTTGGATC 82 76244 76260 125512959 3585 3601 GCTCCGTTGGTGCTTCT 77 n/a n/a 126

TABLE 150 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 % NO: 2 NO: 2 SEQ IDISIS NO Start Site Stop Site Sequence inhibition Start Site Stop Site NO144367 249 268 GGCAGGTCCTTCCTGTGACA 76 21210 21229 11 510701 4217 4233CTCTGTGCTTGGAACTG 78 85147 85163 127 510702 219 235 TGCCTCGATAACTCTGT 7921180 21196 128 561 577 26671 26687 903 919 32218 32234 1245 1261 3776537781 1587 1603 43311 43327 1929 1945 48855 48871 2271 2287 54401 544172613 2629 59956 59972 4299 4315 86472 86488 510704 563 579TGTGCCTCGATAACTCT 80 26673 26689 129 905 921 32220 32236 1247 1263 3776737783 1589 1605 43313 43329 1931 1947 48857 48873 2273 2289 54403 544192615 2631 59958 59974 4301 4317 86474 86490 4985 5001 115495 115511510757 4929 4945 GCTCAGTTGGTGCTGCT 74 n/a n/a 130

Example 118: Dose-Dependent Antisense Inhibition of Apo(a) in TransgenicMouse Primary Hepatocytes

Potent gapmers from the studies described above were further selectedand tested at various doses in transgenic mouse primary hepatocytes in aseries of studies with similar culture conditions. Cells were plated ata density of 35,000 per well and transfected using electroporation with0.156 μM, 0.313 μM, 0.625 μM, 1.250 μM, 2.500 μM, or 5.000 μMconcentrations of antisense oligonucleotide, as specified in the tablesbelow. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and apo(a) mRNA levels were measured byquantitative real-time PCR. Apo(a) primer probe set hAPO(a)12kB was usedto measured mRNA levels. Apo(a) mRNA levels were adjusted according tototal RNA content, as measured by RIBOGREEN®. Results are presented aspercent inhibition of apo(a), relative to untreated control cells.

The results of each of the studies are depicted in the tables presentedbelow with each study represented in a separate table. The half maximalinhibitory concentration (IC₅₀) of each oligonucleotide is alsopresented in the tables.

TABLE 151 0.156 0.312 0.625 1.250 2.500 5.000 IC₅₀ ISIS No μM μM μM μMμM μM (μM) 144367 28 55 70 83 90 92 0.31 510542 33 58 75 87 89 90 0.27510543 33 45 68 78 89 89 0.34 510544 33 50 65 78 88 90 0.33 510545 33 5876 87 91 90 0.26 510546 39 62 76 87 89 91 0.22 510547 36 66 82 84 86 910.22 510548 50 70 82 91 88 90 0.13 510549 32 59 73 85 86 90 0.27 51059526 57 78 88 90 90 0.29 510597 30 53 76 85 89 89 0.30

TABLE 152 0.156 0.312 0.625 1.250 2.500 5.000 IC₅₀ ISIS No μM μM μM μMμM μM (μM) 144367 36 52 78 87 93 94 0.26 510598 48 58 81 88 93 92 0.18510701 45 59 78 87 95 95 0.18 510702 49 63 75 90 94 95 0.15 510704 55 6780 93 94 95 <0.16 510757 34 48 68 79 90 93 0.33 510783 21 32 51 58 78 840.69 512944 57 72 81 91 96 97 <0.16 512947 64 74 86 92 96 97 <0.16512958 48 69 83 91 96 97 0.13 512959 39 59 76 84 93 93 0.22

TABLE 153 0.156 0.312 0.625 1.250 2.500 5.000 IC₅₀ ISIS No μM μM μM μMμM μM (μM) 144367 41 58 75 81 88 87 0.22 510542 38 54 69 74 85 83 0.27510545 21 43 73 77 80 78 0.39 510546 37 58 73 81 83 81 0.24 510547 38 5872 79 84 86 0.24 510548 40 63 77 79 81 84 0.21 510549 37 47 67 77 81 830.31 510595 34 66 73 81 80 75 0.23 510597 39 59 74 83 76 77 0.23

TABLE 154 0.156 0.312 0.625 1.250 2.500 5.000 IC₅₀ ISIS No μM μM μM μMμM μM (μM) 144367 33 60 72 83 81 81 0.26 510598 47 62 75 75 76 76 0.18510701 41 67 80 87 92 91 0.19 510702 51 64 77 80 80 83 0.13 510704 54 6177 84 89 80 0.12 512944 71 74 81 88 92 94 0.02 512947 65 77 86 90 93 950.03 512958 63 73 84 92 93 96 0.06 512959 39 62 80 82 86 82 0.22

Apo(a) mRNA levels were significantly reduced in a dose-dependent mannerin antisense oligonucleotide-treated cells. The potency of the newlydesigned oligonucleotides was compared with the benchmarkoligonucleotide, ISIS 144367. As presented in the tables above, ISIS510542 (SEQ ID NO: 111), ISIS 510545 (SEQ ID NO: 114), ISIS 510546 (SEQID NO: 115), ISIS 510547 (SEQ ID NO: 116), ISIS 510548 (SEQ ID NO: 117),ISIS 510549 (SEQ ID NO: 118), ISIS 510595 (SEQ ID NO: 119), ISIS 510597(SEQ ID NO: 120), ISIS 510598 (SEQ ID NO: 121), ISIS 510701 (SEQ ID NO:127), ISIS 510702 (SEQ ID NO: 128), ISIS 510704 (SEQ ID NO: 129), ISIS512944 (SEQ ID NO: 123), ISIS 512947 (SEQ ID NO: 124), ISIS 512958 (SEQID NO: 125), and ISIS 512959 (SEQ ID NO: 126) were more potent than ISIS144367 (SEQ ID NO: 11).

Example 119: Effect of In Vivo Antisense Inhibition of Human Apo(a) inHuman Apo(a) Transgenic Mice

Transgenic mice with the human apo(a) gene (Frazer, K. A. et al., Nat.Genet. 1995. 9: 424-431) were utilized in the studies described below.ISIS antisense oligonucleotides that demonstrated statisticallysignificant inhibition of apo(a) mRNA in vitro as described above wereevaluated further in this model.

Study 1

Female human apo(a) transgenic mice were maintained on a 12-hourlight/dark cycle and fed ad libitum normal lab chow. The mice weredivided into treatment groups consisting of 4 mice each. The groupsreceived intraperitoneal injections of ISIS 494159, ISIS 494160, ISIS494161, ISIS 494162, ISIS 494163, ISIS 494230, ISIS 494243, ISIS 494244,ISIS 494283, ISIS 494284, ISIS 494285, ISIS 494286, ISIS 494301, ISIS494302, ISIS 494304, ISIS 494466, ISIS 494470, ISIS 494472, ISIS 498239,ISIS 498408, ISIS 498517, ISIS 494158, ISIS 494311, ISIS 494337, ISIS494372, ISIS 498238, ISIS 498523, ISIS 498525, ISIS 510548, ISIS 512944,ISIS 512947, or ISIS 512958 at a dose of 25 mg/kg twice a week for 2weeks. One group of mice received intraperitoneal injections of PBStwice a week for 2 weeks. The PBS group served as the control group. Twodays following the final dose, the mice were euthanized, organsharvested and analyses done.

Inhibition of Human Apo(a) mRNA

Total RNA was extracted from the livers of some of the treatment groups,and human apo(a) mRNA was quantitated by RT-PCR. The results arepresented in the table below, expressed as percent inhibition of apo(a)mRNA compared to the PBS control.

TABLE 155 Percent inhibition of human apo(a) mRNA in transgenic mice %ISIS No inhibition 144367 98 494159 100 494160 95 494161 98 494162 100494163 100 494230 96 494243 99 494244 99 494283 100 494284 100 494285100 494286 98 494301 99 494302 96 494304 94 494466 97 494470 93 49447298 498239 72 498408 100 498517 98

The data demonstrates significant inhibition of apo(a) mRNA by severalISIS oligonucleotides. ISIS 494159 (SEQ ID NO: 14), ISIS 494162 (SEQ IDNO: 17), ISIS 494163 (SEQ ID NO: 18), ISIS 494243 (SEQ ID NO: 106), ISIS494244 (SEQ ID NO: 107), ISIS 494283 (SEQ ID NO: 26), ISIS 494284 (SEQID NO: 27), ISIS 494285 (SEQ ID NO: 28), ISIS 494301 (SEQ ID NO: 39),and ISIS 498408 (SEQ ID NO: 71) were more potent than the benchmark ISIS144367 (SEQ ID NO: 11).

Inhibition of Human Apo(a) Protein

Plasma human apo(a) protein was measured from all treatment groups usingan Apo(a) ELISA kit (Mercodia 10-1106-01, Uppsala, Sweden). The resultsare presented in the table below, expressed as percent inhibition ofapo(a) mRNA compared to the PBS control.

TABLE 156 Percent inhibition of human apo(a) protein in transgenic miceISIS % No inhibition 144367 86 494159 86 494160 0 494161 82 494162 84494163 82 494230 60 494243 84 494244 87 494283 98 494284 98 494285 89494286 89 494301 93 494302 88 494304 83 494466 76 494470 73 494472 72498239 54 498408 84 498517 56 494158 71 494311 83 494337 80 494372 78498238 58 498523 47 498525 58 510548 74 512944 18 512947 65 512958 72

The data demonstrates significant inhibition of apo(a) mRNA by severalISIS oligonucleotides. ISIS 494159 (SEQ ID NO: 14), ISIS 494244 (SEQ IDNO: 82), ISIS 494283 (SEQ ID NO: 26), ISIS 494284 (SEQ ID NO: 27), ISIS494285 (SEQ ID NO: 28), ISIS 494286 (SEQ ID NO: 29), ISIS 494301 (SEQ IDNO: 39), and ISIS 494302 (SEQ ID NO: 40) were as potent as or morepotent than the benchmark ISIS 144367 (SEQ ID NO: 11).

Study 2

ISIS 494159, ISIS 494161, ISIS 494162, ISIS 494163, and ISIS 494243 werefurther evaluated in this transgenic model. ISIS 144367 was included forcomparison.

Treatment

Female human apo(a) transgenic mice were divided into treatment groupsconsisting of 4 mice each. The groups received intraperitonealinjections of ISIS 144367, ISIS 494159, ISIS 494161, ISIS 494162, ISIS494163, or ISIS 494243 at doses of 1.5 mg/kg, 5 mg/kg, 15 mg/kg, or 50mg/kg twice a week for 2 weeks. One group of mice receivedintraperitoneal injections of PBS twice a week for 2 weeks. The PBSgroup served as the control group. Two days following the final dose,the mice were euthanized, organs harvested and analyses done.

Inhibition of Human Apo(a) mRNA

Total RNA was extracted from the livers of the treatment groups, andhuman apo(a) mRNA was quantitated by RT-PCR. The results are presentedin the table below, expressed as percent inhibition of apo(a) mRNAcompared to the PBS control.

TABLE 157 Dose-dependent inhibition of human apo(a) mRNA in transgenicmice Dose % ISIS No (mg/kg/wk) inhibition ED₅₀ 144367 100 71 31 30 42 100 3 5 494159 100 91 5 30 67 10 48 3 39 494161 100 82 6 30 49 10 61 3 30494162 100 90 5 30 67 10 58 3 25 494163 100 83 5 30 66 10 58 3 21 494243100 80 32 30 26 10 0 3 6

The data demonstrates significant inhibition of apo(a) mRNA by severalISIS oligonucleotides. ISIS 494159 (SEQ ID NO: 14), ISIS 494161 (SEQ IDNO: 16), 494162 (SEQ ID NO:17), and ISIS 94163 (SEQ ID NO: 18) were moreefficacious than the benchmark ISIS 144367 (SEQ ID NO: 11).

Reduction of Human Apo(a) Protein Levels

Blood was collected from the treatment groups, and human apo(a) proteinlevels were quantitated by an Apo(a) ELISA kit (Mercodia 10-1106-01,Uppsala, Sweden). The results are presented in the table below,expressed as percent reduction of apo(a) protein levels compared to thePBS control.

TABLE 158 Dose-dependent inhibition of human apo(a) protein intransgenic mice Dose % ISIS No (mg/kg/wk) inhibition ED₅₀ 144367 100 7371 30 0 10 6 3 69 494159 100 88 2 30 88 10 85 3 36 494161 100 90 2 30 8510 73 3 44 494162 100 89 3 30 78 10 76 3 24 494163 100 90 3 30 86 10 603 37 494243 100 61 174 30 0 10 0 3 0

The data demonstrates significant reduction of apo(a) plasma proteinlevels by several ISIS oligonucleotides. ISIS 494159 (SEQ ID NO: 14),ISIS 494161 (SEQ ID NO: 16), ISIS 494162 (SEQ ID NO: 17), and ISIS494163 (SEQ ID NO: 18) were more efficacious than the benchmark ISIS144367 (SEQ ID NO: 11).

Study 3

ISIS 494244, ISIS 494283, and ISIS 494284 were further evaluated in thismodel. ISIS 144367 was included for comparison.

Treatment

Female human apo(a) transgenic mice were divided into treatment groupsconsisting of 4 mice each. The groups received intraperitonealinjections of ISIS 144367, ISIS 494244, ISIS 494283, or ISIS 494284 atdoses of 0.75 mg/kg, 2.5 mg/kg, 7.5 mg/kg, or 25 mg/kg twice a week for2 weeks. One group of mice received intraperitoneal injections of PBStwice a week for 2 weeks. The PBS group served as the control group. Twodays following the final dose, the mice were euthanized, organsharvested and analyses done.

Inhibition of Human Apo(a) mRNA

Total RNA was extracted from the livers of the treatment groups, andhuman apo(a) mRNA was quantitated by RT-PCR. The results are presentedin the table below, expressed as percent inhibition of apo(a) mRNAcompared to the PBS control.

TABLE 159 Dose-dependent inhibition of human apo(a) mRNA in transgenicmice Dose % ISIS No (mg/kg/wk) inhibition ED₅₀ 144367 50 75 22 15 60 5 01.5 0 494244 50 73 18 15 41 5 34 494283 1.5 0 16 50 74 15 52 5 24 1.5 0494284 50 73 16 15 58 5 17 1.5 2

The data demonstrates significant inhibition of apo(a) mRNA by severalISIS oligonucleotides. ISIS 494244 (SEQ ID NO: 107), ISIS 494283 (SEQ IDNO: 26), and ISIS 494284 (SEQ ID NO: 27) were more efficacious than thebenchmark, ISIS 144367 (SEQ ID NO: 11).

Reduction of Human Apo(a) Protein Levels

Blood was collected from the treatment groups, and human apo(a) proteinlevels were quantitated by an Apo(a) ELISA kit (Mercodia 10-1106-01,Uppsala, Sweden). The results are presented in the table below,expressed as percent reduction of apo(a) protein levels compared to thePBS control.

TABLE 160 Dose-dependent inhibition of human apo(a) plasma protein intransgenic mice Dose % ISIS No (mg/kg/wk) inhibition ED₅₀ 144367 50 6416 15 14 5 0 1.5 0 494244 50 67 2 15 60 5 58 1.5 0 494283 50 64 4 15 655 64 1.5 69 494284 50 66 4 15 63 5 51 1.5 54

The data demonstrates significant reduction of apo(a) plasma proteinlevels by several ISIS oligonucleotides. ISIS 494244 (SEQ ID NO: 107),ISIS 494283 (SEQ ID NO: 26), and ISIS 494284 (SEQ ID NO: 27) were moreefficacious than the benchmark, ISIS 144367 (SEQ ID NO: 11).

Study 4

ISIS 494285, ISIS 494286, ISIS 494301, ISIS 494302, and ISIS 494311 werefurther evaluated in this model.

Treatment

Male human apo(a) transgenic mice were divided into treatment groupsconsisting of 4 mice each. Each such group received intraperitonealinjections of ISIS 494285, ISIS 494286, ISIS 494301, ISIS 494302, orISIS 494311 at doses of 5 mg/kg, 15 mg/kg, or 50 mg/kg once a week for 2weeks. One group of 3 mice received intraperitoneal injections of PBSonce a week for 2 weeks. The PBS group served as the control group. Twodays following the final dose, the mice were euthanized, organsharvested and analyses done.

Inhibition of Human Apo(a) mRNA

Total RNA was extracted from the livers of the treatment groups, andhuman apo(a) mRNA was quantitated by RT-PCR. The results are presentedin the table below, expressed as percent inhibition of apo(a) mRNAcompared to the PBS control. The data demonstrates significantinhibition of apo(a) mRNA by ISIS 494285 (SEQ ID NO: 28), ISIS 494286(SEQ ID NO: 29), ISIS 494301 (SEQ ID NO: 39), ISIS 494302 (SEQ ID NO:40) and ISIS 494311 (SEQ ID NO: 47).

TABLE 161 Dose-dependent inhibition of human Apo(a) mRNA in transgenicmice Dose % ISIS No (mg/kg/wk) inhibition ED₅₀ 494285 50 98 1 15 97 5 79494286 50 97 1 15 91 5 80 494301 50 98 3 15 96 5 59 494302 50 98 2 15 885 72 494311 50 99 1 15 96 5 87Reduction of Human Apo(a) Protein Levels

Blood was collected from the treatment groups, and human apo(a) proteinlevels were quantitated by an Apo(a) ELISA kit (Mercodia 10-1106-01,Uppsala, Sweden). The results are presented in the table below,expressed as percent reduction of apo(a) protein levels compared to thePBS control. The data demonstrates significant reduction of apo(a)plasma protein levels by ISIS 494285, ISIS 494286, ISIS 494301, ISIS494302 and ISIS 494311.

TABLE 162 Dose-dependent inhibition of human apo(a) protein intransgenic mice Dose % ISIS No (mg/kg/wk) inhibition ED₅₀ 494285 50 88 215 88 5 72 494286 50 90 2 15 85 5 75 494301 50 89 5 15 86 5 38 494302 5090 3 15 82 5 61 494311 50 90 3 15 82 5 69Study 5

ISIS 494372, ISIS 498524, ISIS 498581, ISIS 498721, and ISIS 498833 werefurther evaluated in this model.

Treatment

Female human apo(a) transgenic mice were divided into treatment groupsconsisting of 4 mice each. The groups received intraperitonealinjections of ISIS 494372, ISIS 498524, ISIS 498581, ISIS 498721, orISIS 498833 at doses of 5 mg/kg, 15 mg/kg, or 50 mg/kg once a week for 2weeks. One group of 3 mice received intraperitoneal injections of PBSonce a week for 2 weeks. The PBS group served as the control group. Twodays following the final dose, the mice were euthanized, organsharvested and analyses done.

Inhibition of Human Apo(a) mRNA

Total RNA was extracted from the livers of the treatment groups, andhuman apo(a) mRNA was quantitated by RT-PCR. The results are presentedin the table below, expressed as percent inhibition of apo(a) mRNAcompared to the PBS control. The data demonstrates significantinhibition of apo(a) mRNA by ISIS 494372 (SEQ ID NO: 28), ISIS 498524(SEQ ID NO: 93), ISIS 498581 (SEQ ID NO: 104), and ISIS 498721(ATGCCTCGATAACTCCGTCC; SEQ ID NO: 134).

TABLE 163 Dose-dependent inhibition of human Apo(a) mRNA in transgenicmice Dose % ISIS No (mg/kg/wk) inhibition ED₅₀ 494372 50 88 18 15 49 5 0498524 50 83 8 15 74 5 34 498581 50 98 7 15 58 5 48 498721 50 97 14 1568 5 0 498833 50 61 155 15 0 5 17Reduction of Human Apo(a) Protein Levels

Blood was collected from the treatment groups, and human apo(a) proteinlevels were quantitated by an Apo(a) ELISA kit (Mercodia 10-1106-01,Uppsala, Sweden). The results are presented in the table below,expressed as percent reduction of apo(a) protein levels compared to thePBS control. The data demonstrates significant reduction of apo(a)plasma protein levels by ISIS 494372 (SEQ ID NO: 28), ISIS 498581 (SEQID NO: 104), and ISIS 498721 (ATGCCTCGATAACTCCGTCC; SEQ ID NO: 134).

TABLE 164 Dose-dependent inhibition of human apo(a) protein intransgenic mice Dose % ISIS No (mg/kg/wk) inhibition ED₅₀ 494372 50 6832 15 25 5 12 498524 50 38 118 15 0 5 0 498581 50 79 9 15 52 5 49 49872150 81 10 15 63 5 29 498833 50 15 738 15 0 5 67

Example 120: Tolerability of Antisense Oligonucleotides Targeting HumanApo(a) in Rodent Models

Gapmer antisense oligonucleotides targeting human apo(a) were selectedfrom the studies described above for tolerability studies in CD1 miceand in Sprague Dawley rats. Rodents do not express endogenous apo(a),hence these studies tested the tolerability of each human antisenseoligonucleotide in an animal rather than any phenotypic changes that maybe caused by inhibiting apo(a) in the animal.

Tolerability in CD1 Mice: Study 1

CD1® mice (Charles River, Mass.) are a multipurpose mice model,frequently utilized for safety and efficacy testing. The mice weretreated with ISIS antisense oligonucleotides selected from studiesdescribed above and evaluated for changes in the levels of variousplasma chemistry markers.

Treatment

Groups of male CD1 mice were injected subcutaneously twice a week for 6weeks with 50 mg/kg of ISIS 494159, ISIS 494161, ISIS 494162, ISIS494244, ISIS 494283, ISIS 494284, ISIS 494285, ISIS 494286, ISIS 494301,ISIS 494302, ISIS 494311, ISIS 494337, ISIS 494372, and ISIS 510548. Onegroup of six-week old male CD1 mice was injected subcutaneously twice aweek for 6 weeks with PBS. Mice were euthanized 48 hours after the lastdose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, albumin,creatinine, and BUN were measured using an automated clinical chemistryanalyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results arepresented in the table below. ISIS oligonucleotides that caused changesin the levels of any of the liver or kidney function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 165 Plasma chemistry markers of CD1 mice ALT AST Albumin BUNCreatinine Bilirubin (IU/L (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) PBS 3871 2.9 25.2 0.16 0.15 ISIS 494159 615 525 2.7 23.9 0.11 0.20 ISIS 494161961 670 2.6 23.7 0.15 0.14 ISIS 494162 1373 1213 2.7 23.7 0.14 0.18 ISIS494283 237 242 2.5 26.2 0.14 0.13 ISIS 494284 192 307 2.3 27.1 0.14 0.10ISIS 494285 582 436 2.3 25.4 0.16 0.11 ISIS 494286 191 227 2.5 21.1 0.120.15 ISIS 494301 119 130 2.7 26.4 0.15 0.12 ISIS 494302 74 96 2.8 24.80.14 0.15 ISIS 494311 817 799 2.7 28.7 0.12 0.17 ISIS 494337 722 397 2.520.0 0.13 0.11 ISIS 494372 73 164 2.6 28.5 0.16 0.11 ISIS 510548 28192245 3.1 26.0 0.15 0.15Organ Weights

Liver, spleen and kidney weights were measured at the end of the study,and are presented in the table below. ISIS oligonucleotides that causedany changes in organ weights outside the expected range for antisenseoligonucleotides were excluded from further studies.

TABLE 166 Organ weights of CD1 mice (g) Kidney Liver Spleen PBS 0.68 2.00.13 ISIS 494159 0.68 3.0 0.21 ISIS 494161 0.62 3.5 0.20 ISIS 4941620.60 3.3 0.20 ISIS 494283 0.65 2.8 0.24 ISIS 494284 0.69 2.7 0.29 ISIS494285 0.59 3.2 0.21 ISIS 494286 0.64 2.8 0.25 ISIS 494301 0.72 3.0 0.43ISIS 494302 0.63 2.3 0.23 ISIS 494311 0.61 3.2 0.19 ISIS 494337 0.56 2.30.17 ISIS 494372 0.60 2.5 0.27 ISIS 510548 0.55 3.7 0.20Tolerability in Sprague Dawley Rats

Sprague-Dawley rats are a multipurpose model used for safety andefficacy evaluations. The rats were treated with ISIS antisenseoligonucleotides selected from studies described above and evaluated forchanges in the levels of various plasma chemistry markers.

Treatment

Groups of male Sprague Dawley rats were injected subcutaneously twice aweek for 8 weeks with 30 mg/kg of ISIS 494159, ISIS 494161, ISIS 494162,ISIS 494244, ISIS 494283, ISIS 494284, ISIS 494285, ISIS 494286, ISIS494301, ISIS 494302, ISIS 494311, ISIS 494337, ISIS 494372, and ISIS510548. One group of six male Sprague Dawley rats was injectedsubcutaneously twice a week for 8 weeks with PBS. Rats were euthanized48 hours after the last dose, and organs and plasma were harvested forfurther analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, albumin,creatinine, and BUN were measured using an automated clinical chemistryanalyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results arepresented in the table below. ISIS oligonucleotides that caused changesin the levels of any of the liver or kidney function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 167 Plasma chemistry markers of Sprague Dawley rats Al- Cre- ALTAST Bilirubin bumin BUN atinine (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL)(mg/dL) PBS 30 82 0.09 3.2 19 0.28 ISIS 494159 182 208 0.14 3.4 22 0.35ISIS 494161 36 86 0.13 3.4 23 0.35 ISIS 494162 102 158 0.17 2.6 28 0.32ISIS 494283 53 156 0.13 2.9 24 0.32 ISIS 494284 34 113 0.08 2.0 28 0.32ISIS 494285 110 294 0.10 1.4 110 0.52 ISIS 494286 40 83 0.07 1.6 48 0.44ISIS 494301 38 132 0.08 3.0 18 0.33 ISIS 494302 47 105 0.09 3.2 19 0.34ISIS 494311 93 185 0.51 2.7 23 0.30 ISIS 494372 54 119 0.12 3.0 19 0.33ISIS 510548 116 181 0.11 1.7 65 0.66Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,urine levels of total protein and creatinine were measured using anautomated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville,N.Y.). Results are presented in the table below, expressed in mg/dL.

TABLE 168 Kidney function markers (mg/dL) in Sprague-Dawley rats TotalCreatinine protein PBS 103 118 ISIS 494159 70 279 ISIS 494161 105 315ISIS 494162 58 925 ISIS 494283 114 1091 ISIS 494284 97 2519 ISIS 49428538 2170 ISIS 494286 51 625 ISIS 494301 62 280 ISIS 494302 101 428 ISIS494311 48 1160 ISIS 494372 46 154 ISIS 510548 55 2119Organ Weights

Liver, spleen and kidney weights were measured at the end of the study,and are presented in the table below. ISIS oligonucleotides that causedany changes in organ weights outside the expected range for antisenseoligonucleotides were excluded from further studies.

TABLE 169 Organ weights of Sprague Dawley rats (g) Kidney liver SpleenPBS 3.5 13.1 0.9 ISIS 494159 3.1 11.7 1.6 ISIS 494161 2.8 12.5 2 ISIS494162 3.1 14.2 1.6 ISIS 494283 3.3 12.9 2.3 ISIS 494284 4.1 15.8 2.7ISIS 494285 3.8 13.4 0.8 ISIS 494286 4.2 16.7 2.5 ISIS 494301 3.2 12.12.3 ISIS 494302 3.4 13.3 2.4 ISIS 494311 3.5 17.4 3.2 ISIS 494372 3.612.9 3.2 ISIS 510548 6.4 21.2 1.5

The finding from the rodent tolerability studies showed that in general,taking into consideration all the tolerability markers screened, ISIS494372 was the best tolerated antisense compound in both the CD1 mousemodel and the Sprague Dawley rat model.

Example 121: Pharmacokinetics of Antisense Oligonucleotide in CD1 Mice

CD1 mice were treated with ISIS oligonucleotides and the oligonucleotideconcentrations in the liver and kidney were evaluated.

Treatment

Groups of four CD1 mice each were injected subcutaneously twice per weekfor 6 weeks with 50 mg/kg of ISIS 494283, ISIS 494284, ISIS 494286, ISIS494301, ISIS 494302, or ISIS 494372. The mice were sacrificed 2 daysfollowing the final dose. Livers were harvested for analysis.

Measurement of Oligonucleotide Concentration

The concentration of the total oligonucleotide concentration wasmeasured. The method used is a modification of previously publishedmethods (Leeds et al., 1996; Geary et al., 1999) which consist of aphenol-chloroform (liquid-liquid) extraction followed by a solid phaseextraction. An internal standard (ISIS 355868, a 27-mer2′-O-methoxyethyl modified phosphorothioate oligonucleotide,GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 131) wasadded prior to extraction. Tissue sample concentrations were calculatedusing calibration curves, with a lower limit of quantitation (LLOQ) ofapproximately 1.14 μg/g. Half-lives were then calculated using WinNonlinsoftware (PHARSIGHT).

The results are presented in the table below, expressed as μg/g liver orkidney tissue. The data indicates that ISIS 494372 was at an acceptableconcentration in the liver and kidneys.

TABLE 170 Oligonucleotide concentration (μg/g tissue) of ISISoligonucleotides in CD1 mice ISIS No Liver Kidney 494283 581 549 494284511 678 494286 368 445 494301 812 347 494302 617 263 494372 875 516

Example 122: Pharmacokinetics of Antisense Oligonucleotide in SpragueDawley Rats

Male Sprague Dawley rats were treated with ISIS oligonucleotides and theoligonucleotide concentrations in the liver and kidney were evaluated.

Treatment

Groups of four rats each were injected subcutaneously twice per week for3 weeks with 10 mg/kg of ISIS 494283, ISIS 494284, ISIS 494286, ISIS494301, ISIS 494302, or ISIS 494372. The rats were sacrificed 2 daysfollowing the final dose. Livers were harvested for analysis.

Measurement of Oligonucleotide Concentration

The concentration of the total oligonucleotide concentration wasmeasured. The method used is a modification of previously publishedmethods (Leeds et al., 1996; Geary et al., 1999) which consist of aphenol-chloroform (liquid-liquid) extraction followed by a solid phaseextraction. An internal standard (ISIS 355868, a 27-mer2′-O-methoxyethyl modified phosphorothioate oligonucleotide,GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 131) wasadded prior to extraction. Tissue sample concentrations were calculatedusing calibration curves, with a lower limit of quantitation (LLOQ) ofapproximately 1.14 μg/g. Half-lives were then calculated using WinNonlinsoftware (PHARSIGHT).

The results are presented in the table below, expressed as μg/g liver orkidney tissue. The data indicates that ISIS 494372 was at an acceptableconcentration in the liver and kidneys.

TABLE 171 Oligonucleotide concentration (μg/g tissue) of ISISoligonucleotides in Sprague Dawley rats ISIS No Liver Kidney 494283 220434 494284 178 573 494286 234 448 494301 279 540 494302 205 387 494372288 663

Example 123: Effect of ISIS Antisense Oligonucleotides Targeting HumanApo(a) in Cynomolgus Monkeys

Cynomolgus monkeys were treated with ISIS antisense oligonucleotidesselected from studies described above. At the time this study wasundertaken, the cynomolgus monkey genomic sequence was not available inthe National Center for Biotechnology Information (NCBI) database;therefore, cross-reactivity with the cynomolgus monkey gene sequencecould not be confirmed. Instead, the sequences of the ISIS antisenseoligonucleotides used in the cynomolgus monkeys was compared to a rhesusmonkey sequence for homology. It is expected that ISIS oligonucleotideswith homology to the rhesus monkey sequence are fully cross-reactivewith the cynomolgus monkey sequence as well.

The human antisense oligonucleotides tested are also cross-reactive withthe rhesus mRNA sequence (XM_001098061.1; designated herein as SEQ IDNO: 132). The greater the complementarity between the humanoligonucleotide and the rhesus monkey sequence, the more likely thehuman oligonucleotide can cross-react with the rhesus monkey sequence.The start and stop sites of each oligonucleotide to SEQ ID NO: 132 ispresented in the table below. Each antisense oligonucleotide targetsmore than one region in SEQ ID NO:132 and has multiple start sites.“Start site” indicates the 5′-most nucleotide to which the gapmer istargeted in the rhesus monkey sequence. ‘Mismatches’ indicates thenumber of nucleotides mismatched between the human oligonucleotidesequence and the rhesus sequence.

Antisense oligonucleotide tolerability, as well as their pharmacokineticprofile in the liver and kidney, was evaluated.

TABLE 172 Antisense oligonucleotides complementary to SEQ ID NO: 132ISIS No Start Site Mismatches 494283 278 2 620 2 923 2 1265 2 1607 11949 1 2267 1 2609 1 2951 1 3293 1 494284 279 1 621 1 924 1 1266 1 16081 1950 1 2268 1 2610 1 2952 1 3294 1 494286 281 1 623 1 926 1 1268 11610 2 1952 2 2270 2 2612 2 2954 2 3296 2 494301 322 2 664 2 967 2 13091 1651 2 494302 323 2 968 2 1310 1 1652 2 494372 1186 2 1870 1 2188 1Treatment

Prior to the study, the monkeys were kept in quarantine for at least a30-day period, during which the animals were observed daily for generalhealth. The monkeys were 2-4 years old and weighed between 2 and 4 kg.Seven groups of four randomly assigned male cynomolgus monkeys each wereinjected subcutaneously with ISIS oligonucleotide or PBS using astainless steel dosing needle and syringe of appropriate size into theone of four sites on the back of the monkeys. The injections were givenin clock-wise rotation; one site per dosing. The monkeys were dosed fourtimes a week for the first week (days 1, 3, 5, and 7) as loading doses,and subsequently once a week for weeks 2-12, with 40 mg/kg of ISIS494283, ISIS 494284, ISIS 494286, ISIS 494301, ISIS 494302, or ISIS494372. A control group of 8 cynomolgus monkeys was injected with PBSsubcutaneously thrice four times a week for the first week (days 1, 3,5, and 7), and subsequently once a week for weeks 2-12.

During the study period, the monkeys were observed at least once dailyfor signs of illness or distress. Any animal experiencing more thanmomentary or slight pain or distress due to the treatment, injury orillness was treated by the veterinary staff with approved analgesics oragents to relieve the pain after consultation with the Study Director.Any animal in poor health or in a possible moribund condition wasidentified for further monitoring and possible euthanasia. For instance,one animal in the treatment group of ISIS 494302 was found moribund onday 56 and was euthanized. Scheduled euthanasia of the animals wasconducted on days 86 and 87 by exsanguination under deep anesthesia. Theprotocols described in the Example were approved by the InstitutionalAnimal Care and Use Committee (IACUC).

Target Reduction

RNA Analysis

On day 86, RNA was extracted from liver tissue for real-time PCRanalysis of apo(a) using human primer probe set ABI Hs00916691_m1(Applied Biosystems, Carlsbad Calif.). Results are presented as percentinhibition of apo(a) mRNA, relative to PBS control. As shown in thetable below, treatment with ISIS antisense oligonucleotides resulted insignificant reduction of apo(a) mRNA in comparison to the PBS control.

The mRNA levels of plasminogen, another kringle-containing protein, werealso measured.

Treatment with ISIS 494372 did not alter the mRNA levels of plasminogen.

TABLE 173 Percent Inhibition of apo(a) mRNA in the cynomolgus monkeyliver relative to the PBS control ISIS No % inhibition 494283 91 49428499 494286 96 494301 88 494302 89 494372 93Protein Analysis

On different days, one mL of blood was collected from the cephalic,saphenous, or femoral vein of all study monkeys. The blood samples wereput into tubes containing K2-EDTA for plasma separation. The tubes werecentrifuged at 3,000 rpm for 10 min at room temperature to obtainplasma. Apo(a) protein levels were analyzed by an Apo(a) ELISA kit(Mercodia 10-1106-01, Uppsala, Sweden). Results are presented aspercentage change of levels from the baseline. As shown in the tablebelow, treatment with several ISIS antisense oligonucleotides resultedin significant reduction of apo(a) protein levels in comparison to thePBS control. Specifically, treatment with ISIS 494372 reducedcynomolgous plasma protein levels of apo(a).

The protein levels of apoB were also measured in the study groups.Antisense inhibition of apo(a) had no effect on apoB levels.

TABLE 174 Apo(a) plasma protein levels (% inhibition over baselinevalues) in the cynomolgus monkey Day 16 Day 30 Day 44 Day 56 Day 72 Day86 PBS 0 0 10 0 0 0 ISIS 494283 78 79 81 66 66 70 ISIS 494284 92 95 9593 93 94 ISIS 494286 92 95 96 94 94 94 ISIS 494301 41 45 52 20 17 29ISIS 494302 17 0 2 0 0 20 ISIS 494372 67 80 83 79 78 81Tolerability StudiesBody and Organ Weight Measurements

To evaluate the effect of ISIS oligonucleotides on the overall health ofthe animals, body and organ weights were measured at day 86. Bodyweights were measured and are presented in the table below. Organweights were measured and the data is presented in the table below. Theresults indicate that treatment with ISIS 494372 was well tolerated interms of the body and organ weights of the monkeys.

TABLE 175 Body weights (g) in the cynomolgus monkey Day 14 Day 35 Day 49Day 56 Day 70 Day 84 PBS 2637 2691 2748 2733 2739 2779 ISIS 494283 25912670 2698 2656 2704 2701 ISIS 494284 2559 2661 2676 2675 2662 2646 ISIS494286 2693 2770 2838 2800 2796 2816 ISIS 494301 2587 2604 2627 25912596 2604 ISIS 494302 2759 2760 2839 2825 3113 3122 ISIS 494372 27192877 2985 2997 3037 3036

TABLE 176 Organ weights (% body weight) in the cynomolgus monkey SpleenKidneys Liver Heart Lungs PBS 0.14 0.38 2.2 0.33 0.51 ISIS 494283 0.240.95 2.8 0.33 0.49 ISIS 494284 0.19 0.60 2.6 0.36 0.55 ISIS 494286 0.220.63 2.7 0.38 0.55 ISIS 494301 0.38 0.81 3.0 0.36 0.61 ISIS 494302 0.170.95 2.5 0.39 0.57 ISIS 494372 0.18 1.16 2.6 0.36 0.56Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,monkeys were fasted overnight prior to blood collection. Approximately1.5 mL of blood was collected from each animal and put into tubeswithout anticoagulant for serum separation. The tubes were kept at roomtemperature for a minimum of 90 min and then centrifuged at 3,000 rpmfor 10 min at room temperature to obtain serum. Levels of various liverfunction markers were measured using a Toshiba 200FR NEO chemistryanalyzer (Toshiba Co., Japan). Plasma levels of ALT and AST weremeasured and the results are presented in the table below, expressed inIU/L. Bilirubin, a liver function marker, was similarly measured and ispresented in the table below, expressed in mg/dL. The results indicatethat treatment with ISIS 494372 was well tolerated in terms of the liverfunction in monkeys.

TABLE 177 Liver function markers in cynomolgus monkey plasma ALT ASTBilirubin (IU/L) (IU/L) (mg/dL) PBS 33 43 0.20 ISIS 494283 75 73 0.12ISIS 494284 115 79 0.17 ISIS 494286 67 73 0.13 ISIS 494301 129 90 0.15ISIS 494302 141 75 0.15 ISIS 494372 46 75 0.17C-Reactive Protein Level Analysis

To evaluate any inflammatory effect of ISIS oligonucleotides incynomolgus monkeys, blood samples were taken for analysis. The monkeyswere fasted overnight prior to blood collection. Approximately 1.5 mL ofblood was collected from each animal and put into tubes withoutanticoagulant for serum separation. The tubes were kept at roomtemperature for a minimum of 90 min and then centrifuged at 3,000 rpmfor 10 min at room temperature to obtain serum. C-reactive protein(CRP), which is synthesized in the liver and which serves as a marker ofinflammation, was measured using a Toshiba 200FR NEO chemistry analyzer(Toshiba Co., Japan). The results indicate that treatment with ISIS494372 did not cause any inflammation in monkeys.

TABLE 178 C-reactive protein levels (mg/L) in cynomolgus monkey plasmaCRP PBS 1.4 ISIS 494283 14.7 ISIS 494284 7.7 ISIS 494286 4.4 ISIS 4943013.5 ISIS 494302 2.4 ISIS 494372 10.2Complement C3 Analysis

To evaluate any effect of ISIS oligonucleotides on the complementpathway in cynomolgus monkeys, blood samples were taken for analysis onday 84 (pre-dose) and day 85 (24 hours post-dose). Approximately 0.5 mLof blood was collected from each animal and put into tubes withoutanticoagulant for serum separation. The tubes were kept at roomtemperature for a minimum of 90 min and then centrifuged at 3,000 rpmfor 10 min at room temperature to obtain serum. C3 was measured using aToshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). The resultsindicate that treatment with ISIS 494372 did not cause any effect on thecomplement pathway in monkeys.

TABLE 179 Complement C3 levels (mg/dL) in cynomolgus monkey plasmaPre-dose Post-dose PBS 140 139 ISIS 494283 127 101 ISIS 494284 105 75ISIS 494286 84 38 ISIS 494301 118 76 ISIS 494302 98 58 ISIS 494372 123109Hematology

To evaluate any effect of ISIS oligonucleotides in cynomolgus monkeys onhematologic parameters, blood samples of approximately 0.5 mL of bloodwas collected on day 87 from each of the available study animals intubes containing K₂-EDTA. Samples were analyzed for red blood cell (RBC)count, white blood cells (WBC) count, as well as for platelet count,using an ADVIA120 hematology analyzer (Bayer, USA). The data ispresented in the table below.

The data indicate that treatment with ISIS 494372 was well tolerated interms of the hematologic parameters of the monkeys.

TABLE 180 Blood cell counts in cynomolgus monkeys WBC RBC Platelet(×10³/μL) (×10⁶/μL) (×10³/μL) PBS 15 6.3 329 ISIS 494283 16 5.3 456 ISIS494284 13 6.3 330 ISIS 494286 14 5.5 304 ISIS 494301 15 6.0 392 ISIS494302 12 6.3 305 ISIS 494372 11 6.1 447

Example 124: Characterization of the Pharmacological Activity of ISIS494372 in Cynomolgus Monkeys

The pharmacological activity of ISIS 494372 was characterized bymeasuring liver apo(a) mRNA and plasma apo(a) levels in monkeysadministered the compound over 13 weeks and allowed to recover foranother 13 weeks.

Treatment

Five groups of 14 randomly assigned male and female cynomolgus monkeyseach were injected subcutaneously with ISIS oligonucleotide or PBS usinga stainless steel dosing needle and syringe of appropriate size into theone of four sites on the back (scapular region) of the monkeys. Themonkeys were dosed four times a week for the first week (days 1, 3, 5,and 7) as loading doses, and subsequently once a week for weeks 2-13 asmaintenance doses, as shown in the table below. The loading dose duringthe first week is expressed as mg/kg/dose, while the maintenance doseson weeks 2-13 are expressed as mg/kg/week.

TABLE 181 Dosing groups in cynomolgus monkeys Test Number of animals fornecropsy Group Article Dose Interim Terminal Recovery 1 PBS — 4 6 4 2ISIS 4 — 6 — 3 494372 8 — 6 — 4 12 4 6 4 5 40 4 6 4

Liver samples from animals were taken at the interim, terminal andrecovery phases of the study for the analyses of apo(a) mRNA. Inaddition, plasma samples were collected on different days to measureapo(a) protein levels. This non-clinical study was conducted inaccordance with the United States Food and Drug Administration (FDA)Good Laboratory Practice (GLP) Regulations, 21 CFR Part 58.

RNA Analysis

Liver samples were collected from monkeys on days 30, 93, and 182, andfrozen. Briefly, a piece (0.2 g) of frozen liver was homogenized in 2 mLof RLT solution (Qiagen). The resulting lysate was applied to QiagenRNeasy mini columns. After purification and quantification, the tissueswere subjected to RT-PCR analysis. The Perkin-Elmer ABI Prism 7700Sequence Detection System, which uses real-time fluorescent RT-PCRdetection, was used to quantify apo(a) mRNA. The assay is based on atarget-specific probe labeled with fluorescent reporter and quencherdyes at opposite ends. The probe was hydrolyzed through the5′-exonuclease activity of Taq DNA polymerase, leading to an increasingfluorescence emission of the reporter dye that can be detected duringthe reaction. A probe set (ABI Rhesus LPA probe set ID Rh02789275_m1,Applied Biosystems, Carlsbad Calif.) targeting position 1512 of therhesus monkey apo(a) mRNA transcript GENBANK Accession No XM_001098061.2(SEQ ID NO: 132) sequence was used to measure cynomolgus monkey liverapo(a) mRNA expression levels. Apo(a) expression was normalized usingRIBOGREEN®. Results are presented as percent inhibition of apo(a) mRNA,relative to PBS control.

As shown in the table below, treatment with ISIS 494372 resulted in adose-dependent reduction of apo(a) mRNA in comparison to the PBScontrol. At day 30, hepatic apo(a) mRNA expression was reduced in adose-dependent manner by 74% and 99% in the 12 mg/kg/week and 40mg/kg/week dosing cohorts, respectively. These reductions arestatistically significant by one-way ANOVA (Dunnett's multiplecomparison test, P<0.05).

Apo(a) mRNA levels were also measured during the recovery phase. Liverexpression levels at day 88 after the last dose were still reduced 49%and 69% in the 12 mg/kg/week and 40 mg/kg/week dosing cohorts,respectively.

TABLE 182 Percent inhibition levels of liver apo(a) mRNA in the dosingphase in cynomolgus monkeys treated with ISIS 494372 Dose Day (mg/kg/wk)% inhibition 30 12 73 40 99 93 4 44 8 43 12 53 40 93Protein Analysis

Approximately 20 μl of plasma was analyzed using a commerciallyavailable apo(a) ELISA kit (Mercodia 10-1106-01, Uppsala, Sweden). Theassay protocol was performed as described by the manufacturer. Theresults are presented in the tables below as percentage change from Day1 pre-dose apo(a) plasma protein concentrations. Statisticallysignificant differences from Day 1 baseline plasma apo(a) using theDunnett's multicomparison test are marked with an asterisk.

Maximal reduction in plasma apo(a) protein was observed in all dosingcohorts by Day 93. In the recovery phase, apo(a) plasma protein levelsin the 40 mg/kg/week dosing cohort were at 22% and 93% of the baselineafter 4 and 13 weeks (Days 121 and 182) of recovery, respectively. Therate of recovery in the 12 mg/kg/week cohort was similar to that seen inthe 40 mg/kg/week cohort.

TABLE 183 Apo(a) plasma protein levels as a percent of Day 1 levels inthe dosing phase in cynomolgus monkeys treated with ISIS 494372 Dose Day(mg/kg/wk) % 30 4 93 8 70 12 49 40  15* 93 4 73 8 56 12  32* 40  11*

TABLE 184 Apo(a) plasma protein levels as a percent of Day 1 levels inthe recovery phase in cynomolgus monkeys treated with ISIS 494372 DoseDay (mg/kg/wk) % 121 12  38* 40  22* 182 12 84 40 93

Example 125: Measurement of Viscosity of ISIS Antisense OligonucleotidesTargeting Human Apo(a)

The viscosity of select antisense oligonucleotides from the studiesdescribed above was measured with the aim of screening out antisenseoligonucleotides which have a viscosity more than 40 centipoise (cP).Oligonucleotides having a viscosity greater than 40 cP would have lessthan optimal viscosity.

ISIS oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μLof water was added and the antisense oligonucleotide was dissolved intosolution by heating the vial at 50° C. Part (75 μL) of the pre-heatedsample was pipetted to a micro-viscometer (Cambridge). The temperatureof the micro-viscometter was set to 25° C. and the viscosity of thesample was measured. Another part (20 μL) of the pre-heated sample waspipetted into 10 mL of water for UV reading at 260 nM at 85° C. (Cary UVinstrument). The results are presented in the table below and indicatethat most of the antisense oligonucleotides solutions are optimal intheir viscosity under the criterion stated above. Those that were notoptimal are marked as ‘viscous’. Specifically, ISIS 494372 was optimalin its viscosity under the criterion stated above.

TABLE 185 Viscosity and concentration of ISIS antisense oligonucleotidestargeting human Apo(a) Viscosity Concentration ISIS No Motif (cP)(mg/mL) 494158 5-10-5 MOE 9.0 350 494159 5-10-5 MOE 11.7 325 4941615-10-5 MOE 12.0 350 494162 5-10-5 MOE 25.8 350 494163 5-10-5 MOE Viscous275 494243 5-10-5 MOE 28.4 325 494244 5-10-5 MOE 19.2 300 494283 3-10-4MOE 13.4 300 494284 5-10-5 MOE 13.4 350 494285 5-10-5 MOE 23.1 350494286 5-10-5 MOE 16.5 275 494301 5-10-5 MOE 17.1 325 494302 5-10-5 MOE24.3 350 494304 5-10-5 MOE 49.3 275 494311 5-10-5 MOE 10.8 325 4943375-10-5 MOE 29.5 325 494372 5-10-5 MOE 12.5 350 494466 5-10-5 MOE Viscous275 494470 5-10-5 MOE 16.7 350 494472 5-10-5 MOE 23.6 350 498408 5-10-5MOE 31.5 300 510548 5-10-5 MOE 9.0 350 512947 3-10-4 MOE 6.8 350 5129585-10-5 MOE 26.0 350

The invention claimed is:
 1. A method comprising administering to ananimal a pharmaceutical composition comprising: a compound comprising asingle stranded modified oligonucleotide and a conjugate group, whereinthe modified oligonucleotide consists of 12 to 30 linked nucleosides andcomprises a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 3901 to 3920 of SEQ ID NO: 1, wherein the nucleobasesequence of the modified oligonucleotide is at least 80% complementaryto SEQ ID NO: 1; wherein the conjugate group is linked to the modifiedoligonucleotide at the 5′ end of the modified oligonucleotide by aconjugate linker; and wherein the conjugate group comprises:

wherein each n is, independently, an integer from 1 to 20; and apharmaceutically acceptable carrier or diluent.
 2. The method of claim1, wherein the animal is a human.
 3. The method of claim 1, for use intreating, preventing, or slowing progression of a disease related toelevated Apo(a) and/or elevated Lp(a).
 4. The method of claim 3, whereinthe disease is a cardiovascular, metabolic and/or inflammatory disease,disorder or condition.
 5. The method of claim 1, wherein the disease ishyperlipidemia.
 6. The method of claim 1, wherein the disease isaneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular disease,coronary artery disease, coronary heart disease myocardial infarction,peripheral vascular disease, peripheral artery disease, peripheralartery occlusive disease, retinal vascular occlusion, or stroke.